The Maternal Immune Activation Mouse Model of Autism Spectrum Disorders
by
Cong Yang (Ingrid) Xuan
A thesis submitted in conformity with the requirements for the degree of Master of Science
Graduate Department of Pharmaceutical Sciences University of Toronto
© Copyright by Cong Yang (Ingrid) Xuan, 2013
ii
The Maternal Immune Activation Mouse Model of Autism
Spectrum Disorders
Cong Yang (Ingrid) Xuan
Master of Science
Department of Pharmaceutical Sciences
University of Toronto
2013
Abstract
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments
in social interaction and communication as well as ritualistic repetitive behaviors.
Epidemiological studies suggest that maternal immune activation (MIA) during pregnancy may
be a risk factor for ASD. To study MIA in a laboratory setting, we injected mouse dams
(C57BL/6) with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (Poly IC) during
mid-gestation to mimic a bacterial or viral infection, respectively. We also performed the same
Poly IC treatment on a mouse model of Fragile X syndrome (i.e. Fmr1 knockout), a genetic
disease with high incidences of ASD. We found modest female-specific impairments in social
interaction and striking male-specific increases in repetitive behavior in adult MIA offspring.
Moreover, prenatal Poly IC treatment caused genotype-specific deficits in sociability in addition
to reduced body weight and rearing in Fmr1 knockout mice only. Therefore, ASD-related
behaviors caused by MIA may be sex, treatment, and/or genotype-dependent.
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Acknowledgments
I would like to express my deepest appreciation for my supervisor Dr. David Hampson for his
helpful guidance and support during this project as well as all the current and past lab members.
I would like to thank my committee members, Dr. Peter Wells and Dr. Paul Frankland for
sharing their expertise during my committee meetings. Lastly, I am extremely grateful to Dr.
Ramsey and her lab members for their generosity with the behavior equipment.
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Table of Contents
Acknowledgments .......................................................................................................................... iii
Table of Contents ........................................................................................................................... iv
List of Figures ................................................................................................................................ vi
List of Appendices ........................................................................................................................ vii
List of Abbreviations ................................................................................................................... viii
1 Introduction ................................................................................................................................ 1
1.1 Autism Spectrum Disorder in Human ................................................................................. 1
1.2 Etiology of ASD: Genetic Makeup ..................................................................................... 6
1.2.1 Fragile X Syndrome ................................................................................................ 7
1.3 Etiology of ASD: Immune Activation ................................................................................ 7
1.3.1 Evidence Based on Epidemiological Studies .......................................................... 7
1.3.2 Evidence Based on Human Pathological and Biochemical Studies ....................... 8
1.4 Maternal Immune Activation: Possible Mechanism in ASD Patients .............................. 10
1.5 Modeling ASD in Rodents Based on the MIA Hypothesis .............................................. 11
2 Rational, Hypotheses and Objectives ....................................................................................... 14
2.1 Rational for MIA Mouse Model ....................................................................................... 14
2.2 Hypotheses and Objectives ............................................................................................... 14
3 Methods .................................................................................................................................... 17
3.1 Animals ............................................................................................................................. 17
3.2 Motor Activity .................................................................................................................. 20
3.3 Social Interaction and Social Preference Test .................................................................. 20
3.4 Marble Burying Test ......................................................................................................... 22
3.5 Grooming .......................................................................................................................... 22
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3.6 Western Blot ..................................................................................................................... 23
3.7 Statistical Analysis ............................................................................................................ 24
4 Results ...................................................................................................................................... 25
4.1 The Effect of MIA on Body Weight ................................................................................. 25
4.2 The effect of MIA on Motor Activity ............................................................................... 25
4.3 The Effect of MIA on Social Behavior ............................................................................. 28
4.4 The Effect of MIA on Repetitive Behavior Evaluated Using Marble Burying Test ........ 36
4.5 The Effect of MIA on Repetitive Behavior Evaluated by Grooming ............................... 38
4.6 The Effect of MIA on the Adult Cerebellum Evaluated by Western Blot ........................ 38
5 Discussion ................................................................................................................................ 41
5.1 The Effect of MIA on Body Weight ................................................................................. 41
5.2 The Effect of MIA on Motor activity ............................................................................... 42
5.3 The Effect of MIA on Social behavior ............................................................................. 43
5.4 The Effect of MIA on Ritualistic and Repetitive Behavior .............................................. 46
5.5 The Effect of MIA on the Adult Cerebellum .................................................................... 49
5.6 Potential Biological Mechanisms for Sex-Dependent Behavioral Changes ..................... 50
6 Conclusions .............................................................................................................................. 52
7 Future Directions ...................................................................................................................... 53
References ..................................................................................................................................... 55
Appendices .................................................................................................................................... 82
vi
List of Figures
Fig. 1 Injection schedule of MIA dams.. ...................................................................................... 18
Fig. 2 Translating human ASD behaviors to mouse model of ASD.. ........................................... 19
Fig. 3 The schedule of behavioral and biochemical analysis of MIA offspring. .......................... 20
Fig. 4 Body weight of wild-type and Fmr1 knockout MIA offspring on PND46 ....................... 26
Fig. 5 Motor activity of LPS wild-type offspring.. ....................................................................... 27
Fig. 6 Motor activity of Poly IC wild-type offspring .................................................................... 29
Fig. 7 Motor activity of Poly IC Fmr1 knockout offspring.. ........................................................ 30
Fig. 8 Social behavior of LPS treated wild-type MIA offspring evaluated by the modified 3-
chamber paradigm. ........................................................................................................................ 32
Fig. 9 Social behavior of Poly IC treated wild-type MIA offspring evaluated by the modified 3-
chamber paradigm.. ....................................................................................................................... 33
Fig. 10 Social behavior of Poly IC treated Fmr1 knockout MIA offspring evaluated by the
modified 3-chamber paradigm.. .................................................................................................... 35
Fig. 11 Repetitive behavior of wild-type and Fmr1 knockout MIA offspring evaluated by the
marble burying test. ...................................................................................................................... 37
Figure 12 Repetitive behavior of MIA wild-type and Fmr1 knockout MIA offspring evaluated by
grooming behavior ........................................................................................................................ 39
Fig. 13 Summary of the expression of different glial markers in the adult cerebellum of LPS
offspring quantified by western blot ............................................................................................. 40
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List of Appendices
Appendix 1 Comparison between computer and manual scoring of social interaction of LPS
treated wild-type MIA female offspring evaluated by the modified 3-chamber paradigm... ……82
Appendix 2 Summary of behavioral findings in wild-type MIA offspring and Fmr1 knockout
MIA offspring.. ............................................................................................................................. 83
Appendix 3 Summary of literature research regarding Poly IC MIA rodent model……………..85
Appendix 4 Summary of literature research regarding LPS MIA rodent model………………...92
viii
List of Abbreviations
1X: immunologically “naïve” dams that were only injected once with either Poly IC or LPS
2X: dams that have been injected with the same agent prior to second injection of Poly IC or LPS
ADHD: attention deficit hyperactivity disorder
ADI-R: Autistic Diagnostic Interview, Revised
Akt (or PKB): protein kinase B
ASD: autism spectrum disorder
ASSQ: autism spectrum screening questionnaire
CDC: Center for Disease Control
CNS: central nervous system
DSM: Diagnostic and Statistical Manual of Mental Disorder
Fmr1: Fragile X mental retardation 1 gene
FMRP: Fragile X mental retardation protein
FXS: Fragile X syndrome
GABRB3: gamma-aminobutyric acid receptor subunit beta-3
GAD: glutamate decarboxylase
GAPDH: anti-glyceraldehyde 3-phosphate-dehydrogenase
GFAP: Glial fibrillary acidic protein
Iba-1: ionized calcium-binding adapter molecule 1
IL: interleukin
INF-γ: interferon- γ IP-10: Interferon gamma-induced protein 10
KO: knockout
LPS: lipopolysaccharide
mTOR: mammalian target of rapamycin
MAPK: mitogen-activated protein kinase
MBP: myelin basic protein
MCP-1: monocyte chemoattractant protein-1
MeCP2: methyl CpG binding protein 2
MHC1: major histocompatibility complex 1
MIA: maternal immune activation
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MRI: magnetic resonance imaging
mTORC1: mammalian target of rapamycin complex 1
NO: nitric oxide
PCs: Purkinje cells
PND: post-natal day
Poly IC or Poly: Polyinosinic-polycytidylic acid
PPI: prepulse inhibition
RANTES: Regulated on Activation, Normal T cell Expressed and Secreted
RELN: reelin
SH3: SRC homology 3
SHANK1: multiple ankyrin repeat domains
STAT: screening tool for autism in two-year-olds
Tsc: tuberous sclerosis complex
TGF-β: transforming growth factor-β
TLR: toll-like receptor
TNF-α: tumour necrosis factor-α
WT: wild-type
1
1 Introduction
1.1 Autism Spectrum Disorder in Human
Autism spectrum disorder (ASD) is a behaviorally defined neurodevelopmental disorder that is
estimated to affect 1 in 88 children in the United States (Center for Disease Control, 2012). The
5th
edition of Diagnostic and Statistical Manual of Mental Disorder (DSM), released in 2013,
requires four criteria to be met for an ASD diagnosis, they are as follows: 1) early childhood
presentation of the symptoms; 2) impaired social communication and interaction; 3) ritualistic
repetitive behavior; 4) symptoms limit everyday functioning.
With regards to the first criterion, ASD is generally diagnosed before the age of two via two
stages (CDC, 2012). The first stage involves screening for developmental problems around 18
months. This is usually conducted by pediatricians in the form of questionnaires such as
checklist of autism in toddlers, screening tool for autism in two-year-olds, and autism spectrum
screening questionnaire. If problems arise, the child would undergo more comprehensive
evaluations to identify the possible origins of the symptoms. These evaluations include genetic
testing, hearing testing, and toxin screening (CDC, 2010).
Autism is diagnosed four times more frequently in males than in females (National Institute of
Health, 2013). The male bias is even stronger in Asperger syndrome, a form of ASD, where the
ratio of male : female is between 9 : 1 and 11 : 1 (Gillberg et al., 2006). Several theories have
been hypothesized with regards to this phenomenon (Baron-Cohen et al., 2011). The first theory
is the natural sexual dimorphism in the human brain during early development. Infant females
tend to have smaller total brain volume and amygdala size than average corresponding males
(Gilmore et al., 2007, Good et al., 2001). This increase in size is further exacerbated in ASD
patients (Schumann et al., 2004, Schumann et al., 2010). The second theory suggests that the
gender bias is due to genetic predisposition. A small percentage of ASDs is caused by X-linked
mutations. This means females will be naturally protected if the activated X-chromosome does
not have the mutation(s). The third theory suggests that the high prevalence of males in ASD
could be caused by elevated fetal testosterone levels. In typically developing children, autistic
traits measured by Childhood Autism Spectrum Test, Child Autism Spectrum Quotient, and
Quantitative Checklist for Autism in Toddlers were positively associated with fetal testosterone
2
levels (Auyeung et al., 2009, Auyeung et al., 2010). In congenital adrenal hyperplasia, female
patients are exposed to high levels of androgens such as testosterone during most of gestation.
Knickmeyer et al. 2006 found that female patients with congenital adrenal hyperplasia had
higher Autism Spectrum Quotient than controls suggesting that the high fetal testosterone
exposure may be responsible for this outcome. Regardless of the gender-bias in ASD, the core
symptoms are the same for both male and female patients.
One of the core symptoms of ASD is deficits in social-emotional reciprocity, impaired
verbal/nonverbal communication during social interaction, and difficulty developing and
maintaining relationships appropriate for their developmental level. Specific behaviors include
lack of reciprocal interaction, gaze aversion and inability to initiate conversation in social
settings. With regards to repetitive behavior or stereotypies, ASD patients must demonstrate at
least two of the following behaviors: repetitive speech or motor movements, insistence on
sameness, fixation of a particular interest, and hyper- or hypo-reactivity to sensory input
(Tchaconas and Adesman, 2013).
Apart from the core symptoms, there are several additional associated conditions of ASD that are
correlated with each other and vary greatly in their severity. For example, some ASD patients
experience heightened or reduced sensitivity to sensory stimuli such as sound and temperature.
Interestingly, behavioral responses to sensory stimuli, such as electrodermal reactivity to tone
(78 decibels), were able to accurately predict sleeping dysfunction in ASD patients (Reynolds et
al., 2012). Furthermore, Baranek et al., 2006 reported that sensory symptoms were inversely
related to the mental age of ASD patients. The level of impairment for specific symptoms also
varies depending on the individual. For instance, in a recent cohort study conducted with 156
ASD patients between ages of 10 and 14, 55% had intellectual disability (IQ < 70), 17% had
below average intelligence (IQ 70-84), 25% had average intelligence (IQ 85-114), and 3% had
above average intelligence (IQ > 115) (Charman, 2011). Other associated symptoms include
seizure, motor impairment, and increased anxiety (Matson et al., 2007, Maski et al., 2011).
Overall, the behavioral impairments of ASD patients are highly variable and difficult to
delineate. This propelled DSM-V to merge the distinct disorders of ASD (with the exception of
Rett syndrome) and reclassify this disorder based on level of severity.
3
There is some evidence suggesting that the severity of the impairments may be higher in second
born siblings compare to first siblings. Martin et al. 2012 showed in a large study (n > 300) that
second siblings demonstrated a decline in nonverbal and verbal IQ scores and increase in autism
severity via social responsiveness scale. This result was reinforced in a separate study where
later-born siblings of children with ASD were at elevated risk of social impairments (Yoder et
al., 2009). In contrast, the repetitive behavior, measured by Autism Diagnostic Interview-
Revised (ADI-R), was more severe in first born siblings compared to second born siblings
(Reichenberg et al., 2007).
To better understand the origin of behavioral abnormalities, scientists investigated the brains of
ASD patients using magnetic resonance imaging (MRI) and biochemical analysis of post-mortem
brain tissues. The cerebellum in particular, has been consistently found to be implicated in the
neuropathology of ASD. Although this brain region is generally associated with motor
coordination, it is also involved in cognitive functions due to its interconnections with other brain
regions such as the cerebral cortex (Fatemi et al., 2012; Broussard, 2013). In a study with 20
cases where the disease was confined to the cerebellum, patients demonstrated impaired
executive functions, personality changes, language deficits, and difficulties with spatial cognition
(Schmahmann and Sherman, 1998). Similarly, in a study consisting of 27 patients with
cerebellar malformations, 74% of patients presented with some degree of mental retardation.
Specifically, patients with cerebellar vermal agenesis or diffuse cerebellar hypoplasia presented
core ASD symptoms including language deficits, social interaction impairments, and some
repetitive and stereotyped behavior. Interestingly, motor deficits in these patients were less
severe (Tavano et al., 2007). These studies highlight the multitude of brain functions that the
cerebellum is involved in.
Structural MRI analyses of autistic children between ages of two and four revealed significant
increases in whole cerebellum volume and cerebellar white matter volume but no change in gray
matter volume (Courchesne et al., 2001, Sparks et al., 2002). However, this overgrowth subsided
by the time the children reached adolescence (Courchesne et al., 2004). The abnormal brain
growth may be partially responsible for the behavioral impairments observed in ASD patients
since the period of overgrowth coincided with the onset of symptoms. In addition, several
groups demonstrated decreases in the size of vermis lobules VI and VII (Akshoomoff et al.,
2004, Kaufmann et al., 2003, Stanfield et al., 2008). This region is involved in oculomotor
4
functions which may be impaired in autism (Takarae et al., 2004). As well, hypoplasia in these
lobules was found to be correlated with decreased exploration and higher rate of stereotyped
behaviors in ASD patients (Pierce and Courchesne, 2001). Interestingly, diffusion tensor
imaging showed increases in axonal integrity in cerebellar white matter regions between the
fastigium of the fourth ventricle and parts of vermis lobules V and VIII which are in close
proximity to lobule VI and VII respectively. Noriuchi et al. 2010 suggested that this may be a
compensatory mechanism for the functional deficits linked with lobule VI and VII.
To understand the neurobiological basis behind these changes in the cerebellar anatomy, analyses
were conducted on post-mortem brain tissues of ASD patients. One of the most reproducible
findings was significant decreases in the number and/or size of Purkinje cells (PCs) (Ritvo et al.,
1986, Kemper and Bauman, 1993, Bailey et al., 1998, Fatemi et al., 2002). It should be noted
that abnormalities in PCs were not observed in all ASD patients, rather, a subpopulation. Using
stereological method, Whitney et al. 2008 showed that only 3 out of 6 ASD patients presented
lower density of PCs compared to controls. The disparity may be due to the heterogeneity of
patient history, small sample size, and method of tissue staining. In addition, the lack of change
in PC density does not exclude the possible changes in its physiology. In fact, Yip et al., 2007
found a 40% reduction in glutamate decarboxylase (GAD) 67 mRNA in PCs based on 8 adult
autistic cases with matched controls.
PCs are the major GABAergic neurons that send inhibitory projections to the deep cerebellar
nuclei. Physical or functional vulnerability of these cells may result in behavioral abnormalities
associated with the cerebellum. Patients with tuberous sclerosis, a genetic disorder that has high
comorbidity with ASD, demonstrated cerebellar pathology that was positively correlated with the
severity of ASD symptoms (Ertan et al., 2010, Weber et al., 2000, Eluvathingal et al., 2006). In
rodents, loss of tuberous sclerosis complex (Tsc) 1 or Tsc2 in PCs both resulted in PC
degeneration that caused ASD-like behaviors including social impairment, increased repetitive
behavior and abnormal vocalization (Tsai et al., 2012, Reith et al., 2013). Similarly, lurcher
mice, which exhibit depletion of PCs during development, displayed ASD characteristics
including increases in repetitive behavior and deficits during reversal learning tasks (Martin et
al., 2010, Dickson et al., 2010). Therefore, abnormalities in PCs may contribute to ASD
symptomology.
5
In addition to the cerebellum, pathologies in other brain regions may also contribute to the
development of ASD. One such region is the amygdala which is part of the limbic system
responsible for emotion and memory (Phelps, 2006). Similar to the growth progression of the
cerebellum, the amygdala was also found to be enlarged during early childhood compared to
controls but the growth subsided during adolescence and thereafter (Sparks et al., 2002,
Schumann et al., 2004). More importantly, in ASD children between age 3 and 7, this
enlargement was associated with deficits in social and communication skills (Munson et al.,
2006, Kim et al., 2010). In a small study of 9 cases, autistic patients had significantly fewer
neurons in the amygdala than respective controls (Schumann and Amaral, 2006). Moreover,
quantitative MRI revealed that the grey matter volume was significantly increased in the anterior
temporal and dorsolateral prefrontal region and reduced in the occipital and medial parietal
regions of ASD patients compared to controls. These changes were significantly correlated with
the severity of ASD symptoms measured by ADI-R in the social, communication, and/or
repetitive behavior domain (Ecker et al., 2012). To summarize, several regions of the brain are
involved in the pathology of ASD, particularly the cerebellum.
Despite major scientific advances in the field of autism, no cure has currently been identified.
The effectiveness of treatment can be improved with early diagnosis and intervention (Reichow
et al., 2009, Rogers and Vismara, 2008). The primary treatment for ASD is through educational
therapy. These therapies focus on relationship building as well as teaching social and
communication skills. The most widely accepted therapy is the applied behavior analysis (ABA)
which uses small groups to focus on shaping and reinforcing social communication by prompting
and encouraging play time (Couper et al., 2003). Medications used to treat ASD-related
conditions have been found to be effective for some patients. Risperidone and aripiprazole,
which are antipsychotic drugs, were shown to be effective for treating repetitive behaviors in
children with ASDs but also caused significant adverse effects (McPheeters et al., 2011). Nasal
oxytocin spray is currently investigated as an ASD drug to potentially improve social
impairments. Thus far, a small clinical trial of 7 patients showed promising results using
functional MRI studies to evaluate brain patterns associated with social functions (Eisenstein,
2012). Methylphenidate (Ritalin), a psychostimulant that is commonly prescribed for attention
deficit hyperactivity disorder (ADHD), were administered to alleviate hyperactivity in ASD
patients but not all responded to the treatment (Posey et al., 2007). Furthermore, some
6
complementary and alternative medicine therapies have been reported to be beneficial in certain
cases of ASD. Examples include gluten-free diet, omega-3 fatty acid supplements, hyperbaric
oxygen therapy, and chemical chelation. However, the validity of these treatments remains to be
investigated since they were mostly anecdotal (Tchaconas and Adesman, 2013).
Finding a cure is difficult because we still do not know the etiology of this disorder. Due to the
heterogeneous nature of ASD, there is almost certainly more than one possible cause. The most
established theories by the scientific community are genetic makeup and immune
activation/dysregulation (Onore et al., 2012). Below, I will discuss the evidence supporting these
two theories based on human and rodent studies.
1.2 Etiology of ASD: Genetic Makeup
Autism spectrum disorder can be classified as either idiopathic or syndromic. Idiopathic ASD
represents cases where ASD is the primary diagnosis and the cause is unknown. Twin studies in
ASD suggest that the genetic makeup of the individual could provide one explanation for its
etiology. The concordance rate for dizygotic twins was found to range from 0% - 30% whereas
for monozygotic twins, it is increased to 60% - 90% (Steffenburg et al., 1989, Bailey et al., 1995,
Rutter et al., 1999, Taniai et al., 2008, Rosenberg et al., 2009, Lichtenstein et al., 2010, Ronald et
al., 2011). This encouraged more in-depth genetic analysis which revealed several ASD risk
genes including reelin (RELN), SRC homology 3 and multiple ankyrin repeat domains
(SHANK1), and gamma-aminobutyric acid receptor subunit beta-3 (GABRB3) (Buxbaum et al.,
2002, Skaar et al., 2005, Sato et al., 2012). More convincingly, knockout (KO) mice or mice
with mutation(s) in these genes demonstrated ASD-related behaviors and pathologies (DeLorey
et al., 2008, Silverman et al., 2010, Won et al., 2012). Syndromic ASDs are genetic disorders
where ASD is a comorbid condition in addition to a pre-existing genetic mutation(s). Examples
include Rett syndrome, Fragile X syndrome (FXS), and tuberous sclerosis which are caused by
single gene mutations in the methyl CpG binding protein 2 (MeCP2), Fragile X mental
retardation 1 (Fmr1), and tuberous sclerosis complex (Tsc1 or Tsc2) gene, respectively. The
corresponding incidences of ASD in these diseases were estimated to be 25 - 40%, 21 - 50%, and
24 - 60% (Moss et al., 2009). Nevertheless, only 10 - 20% of the ASD population can be
attributed to genetic mutations and risk factors (Abrahams and Geschwind, 2008).
7
1.2.1 Fragile X Syndrome
Fragile X syndrome (FXS) is genetic disorder caused by a single gene mutation in the Fmr1 gene
on the X chromosome. This results in an expansion of the number of CGG trinucleotide repeats
(>200) in the 5’ untranslated region which silences the FMR1 gene via hypermethylation. This
prevents the production of Fragile X mental retardation protein (FMRP) which is an mRNA
binding protein, primarily found in the brain, testes, and ovaries. It is responsible for shuttling
target mRNA to dendritic spines and inhibiting its translation by associating with specific
ribosomes. The lack of this protein leads to perturbations in synaptic plasticity and protein
translation (Bassell and Warren, 2008). FXS patients have characteristic physical phenotypes
including large, protruding ears, long face, and macroorchidism. Behavioral consequences of
this disease heavily overlap with ASD which include mental retardation, stereotypic movements
and abnormal social development (Jin and Warren, 2003). In fact, approximately 2 – 6% of
children with autism have FXS (Estecio et al., 2002, Hagerman and Hagerman, 2002; Harris et
al., 2008; Reddy, 2005, Wassink et al., 2001).
1.3 Etiology of ASD: Immune Activation
1.3.1 Evidence Based on Epidemiological Studies
Compared to twins, the incidence of ASD in siblings was estimated to be only 3 – 8% (Smalley
et al., 1991, Muhle et al., 2004, Lauritsen et al., 2005, Sumi et al., 2006). A recent twin study
found that 58% of heritability in ASD can be explained by environmental factors (Hallmayer et
al., 2011). This suggests that environmental factors such as the maternal environment during
fetal development could contribute to ASD since fraternal twins share the same placenta.
One of the most established environmental factors linked to increasing risk of ASD is infection
during pregnancy. Studies published as early as the 1970s found that the rubella epidemic in
1964 was associated with significant increased incidences of ASD in children (Chess, 1971,
Swisher and Swisher, 1975, Chess et al., 1978). As well, congenital infection of
cytomegalovirus was reported in ASD patients (Sweeten et al., 2004). Perhaps the most
convincing evidence was presented recently by a Danish cohort study of more than 20,000 ASD
patients spanning from 1980 to 2005. In this study, mothers that gave birth to ASD children had
higher incidences of autoimmune disease, such as rheumatoid arthritis, compared to control.
8
More importantly, the risk of autism was increased by 3-fold with maternal viral infection and by
2-fold with maternal bacterial infection (specifically respiratory infection) in the first trimester
(Atladóttir et al., 2010). In a more recent study, the same group reported that out of 976 children
diagnosed with ASD, mothers who had fever lasting more than 7 days during first and second
trimester increased the risk of their baby developing infantile autism by 2.9 and 4.2 fold,
respectively (Atladóttir et al., 2012). These studies support the theory that environmental insults,
such as an infection during pregnancy, could increase the risk of the fetus developing ASD.
1.3.2 Evidence Based on Human Pathological and Biochemical Studies
Within the central nervous system, microglia and astrocytes are activated as part of the innate
immune response during neuroinflammation. Microglia is able to migrate to the site of insult
and clear debris by increasing the rate of phagocytosis and exerting cytotoxicity (Napoli and
Neumann, 2009). In addition, both astrocytes and microglia can activate the adaptive immune
system by triggering cytokine production and recruiting T-cells by expressing major
histocompatibility complex (Kreutzberg, 1996, Hamo et al., 2007). In ASD patients between the
age of 5 and 44, increased activation in microglia and astrocytes was observed in the cerebellum,
middle frontal gyrus, and anterior cingulate gyrus of post-mortem tissues (Vargas et al., 2005).
This result was replicated in a recent study of 20 male ASD patients (age 18 – 31) using positron
emission tomography where they found activation of microglia in brain regions including the
cerebellum, midbrain, pons, fusiform gyri and the anterior cingulate and orbitofrontal cortices
with the cerebellum showing the most prominent increases (Suzuki et al., 2013). In a separate
study of ASD patients of similar age range, microglial somal volume and cell density were
increased in the white matter and gray matter of the dorsolateral prefrontal cortex of ASD
patients (Morgan et al., 2010). The large age range of the population sample suggests that
immune activation in ASD may persist into adulthood.
Cytokines, produced mainly by immune cells, are the pleiotropic mediators between the immune
system and the CNS. They participate in a variety of neurological processes in the CNS such as
progenitor cell differentiation/migration, synaptic network modification and pruning, and cell
signaling cascade induction (Deverman and Patterson, 2009). The cytokines that were most
consistently found to be implicated in ASD pathogenesis are: interleukins-1β (IL-1β),
interleukin-6 (IL-6), interleukin-4 (IL-4), interferon- γ (INF-γ) and transforming growth factor-β
9
(TGF-β) (Goines and Ashwood, 2012). IL-1β and IL-6 are proinflammatory cytokines that were
found to be both elevated in the plasma of ASD children and adults (Ashwood et al., 2011,
Emanuele et al., 2010, Suzuki et al., 2011). In contrast, IL-4, INF-γ and TGF-β serve more
neuroprotective roles in the CNS. IL-4 level was reported to be increased in the serum and
amniotic fluid of mothers with ASD children (Goin et al., 2011, Abdallah et al., 2011). INF-γ
and TGF-β levels were elevated and decreased in the plasma of ASD patients, respectively
(Singh, 1996, Croonenberghs et al., 2002, Vargas et al., 2005, Okada et al., 2007, Ashwood et
al., 2008, Li et al., 2009).
These cytokines activate similar pathways such as the mitogen-activated protein kinase (MAPK)
signalling pathway (Nelms et al., 1999, O’Neill, 2000, Shi and Massague, 2003, Platanias, 2005,
Hsiao et al., 2011). Therefore, it is not surprising that they can elicit similar functions in the
CNS. IL-1β was found to be expressed immediately upon insult and was able to inhibit neural
progenitor cell proliferation and promote gliogenesis (Crampton et al., 2012, Peng et al., 2008).
Furthermore, intracerebral injection of IL-1β was able to decrease the expression of myelin basic
protein (MBP, a marker for mature oligodendrocytes) in neonatal rats (Cai et al., 2004). On the
contrary, microglia activated by IL-4 was able to promote oligodendrogenesis (Butovsky et al.,
2006). Therefore, both cytokines could be implicated in the region-specific growth
abnormalities and white matter alternations found in ASD brains (de la Mano et al., 2007).
Synapses are also influenced by dysregulation of these cytokines. For instance, chronic IL-6
expression was able to shift the ratio of excitatory to inhibitory synapses in cerebellar granular
cell cultures (Wei et al., 2011). As well, INF-γ was able to induce synaptic alternations by
increasing the expression of major histocompatibility complex 1 (MHC1), triggering T cells and
natural killer cells (Shatz, 2009). Furthermore, lack of TGF-β in mice showed deficits in
glutamatergic and GABAergic synapses (Vashlishan et al., 2008). Changes at these synapses
may be involved in the behavioral symptoms found in ASD patients.
Cytokines are multifunctional and work within a network of signaling molecules. They are able
to exert both neuroprotective and neurotoxic responses in the brain. Too much or too little
expression can both be detrimental to proper brain functioning. For instance, both
overexpression and under expression of IL-1β in the hippocampus were associated with impaired
memory and learning (Goshen et al., 2007, Barrientos et al, 2009, Labrousse et al., 2009). The
time that these cytokines are activated is also important. The overexpression of TGF-β1 in the
10
dentate gyrus during development led to deficits in social interaction and increased repetitive
behavior. However, these behavioral characteristics were the opposite when the overexpression
was introduced during adulthood (Depino et al., 2011). In addition, these cytokines are able to
regulate each other. They induce immune responses by interacting closely with glial cells. For
example, IL-1β was found to stimulate astrocyte growth as well as induce the activation of TGF-
β (Ransohoff and Benveniste, 1996). Other cytokines such as tumour necrosis factor-α (TNF-α),
interleukin-10 (IL-10), and monocyte chemoattractant protein-1(MCP-1) were also found to be
involved in ASD (Goines and Ashwood, 2012).
Overall, there is strong evidence that the level of cytokines is altered in maternal serum as well
as in the serum and brain tissue of ASD patients. Changes in these cytokine levels could
contribute to the pathological and behavioral deficits in ASDs. The exact mechanism is still
unclear because responses elicited by cytokines vary depending on the time of activation and the
environmental conditions. Nevertheless, it is clear that the balance of these cytokines is vital for
normal brain functioning.
1.4 Maternal Immune Activation: Possible Mechanism in ASD Patients
The MIA hypothesis is supported by epidemiological studies and the presence of immune
dysregulation observed in ASD patients as described above. The hypothesis suggests that an
infection to the mother during pregnancy can cause the production of various cytokines. The
imbalance of these signaling molecules may trigger the fetal immune system and perturb proper
brain maturation resulting in brain pathologies and ASD-associated behaviors.
The mechanism of how the activation of the maternal immune system causes changes in the fetal
brain is still unclear. As discussed in the previous section, cytokines are the main mediators
upon immune activation. However, most of the cytokines were found to be unable to cross the
placenta, with the exception of IL-6 (Zaretsky et al., 2004, Aaltonen et al., 2005). In fact, in fetal
inflammatory response syndrome, the level of IL-6 was found to be increased in the amniotic
fluid as well as maternal, fetal and umbilical cord serum (Gomez et al., 1998, Madsen-Bouterse
et al., 2010). Furthermore, incubation of human placenta with lipopolysaccharide (LPS), which
stimulated a bacterial infection, produced increased level of IL-6 (Holmlund et al., 2002).
Therefore, it is likely that IL-6 plays a major role in translating the maternal immune activation
11
to the fetus. Smith et al., 2007 explored this hypothesis in rodents and found that injection of
recombinant IL-6 to wild-type mouse dams during mid-gestation resulted in ASD-like behaviors
such as social deficits and decreased prepulse inhibition in the litters. However, this was not
observed in litters from IL-6 knockout dams. More importantly, the behavioral deficits can be
reversed with injection of IL-6 antibody. Therefore, IL-6 signaling could be one of the key
participants in the MIA mechanistic pathway.
1.5 Modeling ASD in Rodents Based on the MIA Hypothesis
Rodent models can be used to further investigate the potential effects of maternal immune
activation on the fetal brain. In this model, pregnant dams were injected with an infectious agent
during pregnancy to activate the maternal immune system. The agents that were most commonly
used are polyinosinic:polycytidylic acid (Poly IC) and LPS which simulate viral and bacterial
infections, respectively. Poly IC is a synthetic double stranded RNA that mimics a viral
infection by binding to toll-like receptor (TLR) 3 while LPS is an endotoxin that mimics a
bacterial infection by binding to TLR4 (Muzio et al., 2000, Alexopoulou et al., 2001, Visintin et
al., 2001, Netea et al., 2002). In humans, both types of TLRs are highly expressed in microglia.
However, only TLR3 is also expressed in astrocytes and oligodendrocytes (Bsibsi et al, 2002;
Farina et al, 2005). The activation of these TLRs on different types of cells could potentiate
different downstream effects. The offspring from these immune activated dams were subjected
to behavioral tests linked to symptoms of ASD. As well, their brain and serum were analyzed to
identify the presence of immune activation and ASD associated neuropathologies.
Thus far, the behavioral and pathological phenotypes of the MIA model, summarized in the
Appendix 3 and 4, look promising. However, it is difficult directly compare these models
because they differ in the specific agent used, route of administration, dose, and injection regime.
The most common route of administration was through intraperitoneal (i.p.) injection. However,
subcutaneous, intravenous and tail vein injections were also performed. The dose is also
important because higher doses may be lethal to the litter or the dam. This was the case with
LPS (serotype: O111:B4) where i.p. injection of 100 μg/kg on E10 and E11 resulted in 0% litter
delivery whereas injection of 50 μg/kg on the same dates resulted in 100% litter delivery (Fortier
et al., 2007). In addition, the serotype of LPS is an important factor because certain serotypes
may be more potent than others. Compare to injection of LPS with serotype of O111:B4 (Fortier
12
et al., 2007), a much higher dose (1 mg/kg) of LPS with serotype of O26:B6 was injected using
the same method and at the same time, but 100% of the litter survived (Ling et al., 2009). The
gestation day(s) and frequency of the injection(s) should be considered as well. LPS or Poly IC
injection(s) during early pregnancy may cause embryonic resorption associated with oxidative
stress. Oxidative stress occurs as a consequence of the body’s inability to properly detoxify
reactive oxygen species such as peroxides and free radicals. Nitric oxide (NO) is a free radical
that can exert numerous toxic effects including inhibition of mitochondrial respiratory chain,
DNA break, apoptosis, and necrosis (Karima et al., 1999). Ogando et al., 2003 found that high
doses of LPS injection (500 μg/kg) during early pregnancy (E7) increased the decidual and
uterine NO production due to increased mRNA expression of inducible and neuronal nitric oxide
synthase. This resulted in 100% embryonic resorption 24 hours post injection. Additional
injections of aminoguanidine, an inducible nitric oxide synthase inhibitor, were able to rescue
this outcome. Similarly, prenatal Poly IC injection on the same gestation day (E7) also increased
embryonic resorption rate to 54.8%. As well, the mRNA expression of iNOS was also increased
in the embryonic unit which consisted of the embryo, placenta and uterine muscle (Shimada et
al., 2003).
The fetal immune system can be induced by activating the maternal immune system. In the LPS
model, the cytokine levels (i.e. IL-1β, IL-6, and/or TNF-α) were elevated in the placenta,
amniotic fluid, fetal serum and fetal brain tissue for multiple serotypes of LPS (Urakubo et al.,
2001, Ashdown et al., 2006, Liverman et al., 2006, Salminen et al., 2008, Ning et al., 2008,
Romero et al., 2010, Girard et al., 2010). For Poly IC model, the increase in IL-6 and MCP-1
was observed in the amniotic fluid and dam serum, respectively (Forrest et al., 2012, Mandal et
al., 2010, 2011). Garay et al. 2012 reported that changes in the levels of different cytokines in
the Poly IC model were dependent on the post-natal age and specific brain regions. For example,
they found that the level of IL-1β in the frontal cortex was significantly elevated at PND0 but
decreased at PND14 and PND30. In comparison, level of IL-1β was significantly reduced in the
hippocampus at PND0 and not changed at later time points. Remarkably, the cytokines that were
changed in the MIA model were also found to be altered in ASD patients. One such example is
IL-1β, which was found to be significantly elevated in the sera of both young mice prenatally
injected with Poly IC and ASD children.
13
In the LPS model, injection(s) during mid- and late-gestation yielded increased in the expression
of astrocyte (Cai et al., 2000, Paintlia et al., 2004, Paintlia et al., 2008) and microglia marker(s)
(Larouche et al., 2005, Ling et al., 2006, Ling et al., 2009, Roumier et al., 2008, Girard et al.,
2010) as well as decreased in the expression of mature oligodendrocyte marker(s) (Cai et al.,
2000, Kumral et al., 2007, Yesilirmak et al, 2007, Paintlia et al., 2004, 2008, Rousset et al., 2006,
2008). For Poly IC, Juckel et al., 2011 and Makinodan et al., 2008 showed mid-gestation
injection (E9 - 9.5) increased microglia activation in the hippocampus and striatum as well as
decreased myelin in the hippocampus. These data mirror the pathologies observed in ASD
patients and support the idea that maternal immune activation is able to elicit brain perturbations
in the fetus.
Ideally, the MIA model for ASD should demonstrate the core symptoms of this disorder.
Malkova et al, 2012 accomplished this using Poly IC mouse model where they showed that male
offspring displayed deficits in social interaction, impaired communication and increased
repetitive behavior. Furthermore, the deficits in social interaction in the Poly IC model were
confirmed by Soumiya et al., 2011. Similarly, several groups have also reported impairments in
social interaction in the LPS model (Golan et al., 2006, Kirsten et al., 2010, 2012, Oskvig et al.,
2012). In addition, both Poly IC and LPS model demonstrated several ASD-associated behaviors
such as increased anxiety in novel environment (Bakos et al., 2004, Smith et al., 2007, de
Miranda et al., 2010, Wang et al., 2010, Soumiya et al., 2011, Hsiao et al, 2011) and deficits in
sensory motor gating (Borrell et al., 2002, Shi et al., 2003, Fortier et al., 2007, Romero et al.,
2007, 2010, Makinodan et al., 2008, de Miranda et al., 2010, Hsiao and Patterson, 2011).
14
2 Rational, Hypotheses and Objectives
2.1 Rational for MIA Mouse Model
We decided to inject wild-type C57/B6 mice with Poly IC (20 mg/kg, i.p.) on E12.5 or LPS
(serotype O127:B8, 75 μg/kg, i.p.) on E11.5 and E12.5. We also performed the same Poly IC
injection on Fmr1 KO C57/B6N mice. The Poly IC model was established based on published
ASD-related behavioral and neuropathological findings using the same injection regime (Smith
et al., 2007, Shi et al., 2009, Mandal et al., 2010, 2011, Hsiao and Patterson, 2011).
Furthermore, the gestation day(s) of the injections coincided with the peak of PC generation in
the cerebellum and amygdala neurogenesis which occurs at 10 – 13 days and 11 – 14 days
respectively (Workman et al., 2013). As previously discussed, these two brain regions were
heavily implicated in ASD. LPS was injected around the same time because we wanted to
directly compare the differences between viral and bacterial infection. The LPS serotype
O127:B8 was used because it was the most common serotype used in mice by other groups.
Using this particular serotype, these groups demonstrated immune activation in amniotic fluid
and/or fetal brain (Liverman et al., 2006, Ning et al., 2008) as well as some behavioral changes
including increased anxiety (Wang et al., 2010) and hyperactivity after acute amphetamine
challenge (Zager et al., 2012).
2.2 Hypotheses and Objectives
We hypothesized that maternal immune activation in mice using Poly IC and LPS during mid-
gestation will elicit ASD-like behaviors and cerebellar pathologies in the offspring that may be
dependent on sex of the offspring and type of prenatal infection (i.e. LPS vs. Poly IC).
Furthermore, the effect of MIA may be exacerbated in offspring from dams with immunological
memory (i.e. Poly IC 2X and LPS 2X) and/or genetic mutation (i.e. Fmr1 KO).
Objective 1: To compare the effects of bacterial (LPS) and viral (Poly IC) prenatal infection in
MIA offspring
Rational: LPS and Poly IC mimic different types of infection in the mother by acting on different
toll like receptors. Thus far, only Harvey and Boksa, 2012 has directly compared these two
models and found prenatal treatment-dependent changes in neuronal density (at PND14) and
15
reelin-positive cells (at PND28) in the stratum oriens of CA1. No other groups have investigated
the behavioral differences between these two models.
Objective 2: To investigate whether male vs. female MIA mice show differential effects in ASD-
related behaviors and cerebellar pathologies
Rational: Because of the strong male-bias in ASD, it would be interesting to investigate if this
phenomenon is also present in the MIA mouse model. Most of the studies published to date
were conducted on male mice only, or a mixture of both sexes. By combining them during the
analysis, it could mask the significance that may be present for only one sex. For example,
Wang et al., 2010 showed that female offspring (PND200) from LPS treated dams had impaired
exploration in open field test whereas male offspring of the same age did not showed such
difference.
Objective 3: To investigate cerebellar pathology in adult MIA offspring
Rational: Human pathological studies suggest that the cerebellum is perturbed in ASD.
However, most groups that investigated neuroinflammation focused on the forebrain during early
development. Our study will focus on specifically investigating the pathological changes in the
adult cerebellum as it pertains to ASD.
Objective 4: To investigate the effects of “immunological memory” of dams on MIA offspring
Rational: All of the studies published to date administered LPS or Poly IC injections on
immunologically “naïve” mice. In contrast to rodents, most pregnant woman would have been
exposed to different pathogens prior to their pregnancy. Therefore, it would be biologically
relevant for human to investigate the MIA model using previously immunized dams. So far,
only Mandal et al., 2011 has investigated the difference in immune response between litters from
naïve and immunized dams injected with Poly IC. They found that although both naïve and
previously immunized dams had comparable serum and amniotic fluid levels of IL-6 before and
after Poly IC injection, only offspring from immunized dams demonstrated differentiation of T-
helper 17 (Th17) cells. These cells are a subset of T helper cells that produce IL-17 which can
contribute to neuroinflammation by recruiting monocytes and neutrophils to the site of
inflammation (Korn et al., 2009). Our goal was to compare the severity of neuropathological
16
and/or behavioral outcomes between litters from immunologically “naïve” dams (i.e. Poly 1X or
LPS 1X) and litters from dams re-injected with Poly IC or LPS (i.e. Poly 2X or LPS 2X).
Objective 5: To investigate the effects of MIA on a genetic model of ASD (i.e. Fmr1 KO mouse)
Rational: There is some evidence of immune dysregulation in Fragile X syndrome. Ashwood et
al., 2010 reported that the serum level of several cytokines (IL-1α, RANTES, and IP-10) in
Fragile X patients with or without ASD was significantly different from typically developing
individuals. In addition, increased GFAP expression was observed in several brain regions
including the cerebellum of adult Fmr1 knockout mice, a possible sign of neuroinflammation
(Yuskaitis et al., 2010, L. K.K. Pacey, I. Xuan, D. R. Hampson, unpublished data). We injected
Poly IC into Fmr1 knockout dams to determine whether the effects of prenatal viral insult could
be exacerbated in offspring with pre-existing genetic vulnerability.
17
3 Methods
3.1 Animals
All animal experiments were carried out in accordance with the guidelines set out by the
Canadian Council on Animal Care and were approved by the University of Toronto Animal Care
Committee. FMR1 knockout mice (backcrossed > 10 generations on the C57BL/6 background)
were generously provided by Dr. William Greenough, University of Illinois, and bred at the
University of Toronto. Only homozygous female Fmr1 knockout mice were used for breeding
therefore genotyping was not performed. Mice were mated overnight and vaginal plug was
checked at 9 am and 4 pm each day. The day that the vaginal plug was observed was marked as
embryonic day 0.5. One group of dams were given intraperitoneal (i.p.) injections (5 μl/g) of 75
μg/kg of LPS (lipopolysaccharide O127:B8; L3129; Sigma) on both E11.5 and E12.5. A
separate group of dams were injected (i.p. 5 μl/g) with 20 mg/kg polyinosinic:polycytidylic acid
(Poly IC) potassium salt (P9582; Sigma) once on E12.5. Poly IC was dissolved in saline at 4
mg/mL (based on the weight of Poly IC itself, which was 10% of total weight of the salt). Saline
injections were made on either E11.5 and E12.5 or E12.5 only, and were used as control. A
schematic of the injection schedule is shown in Fig. 1.
All pups were housed with their mother until post-natal day (PND) 21 when they were weaned
and ear tagged. After weaning, two to four pups of the same sex and treatment were housed
together per cage. In case where only one single female or male was born in one litter, it was
housed with other mice of same sex and similar age (may be of different treatment group). All
animals were left undisturbed except for biweekly cage changes and behavior tests. All
behavioral testing with the exception of social test was conducted between 9 am and 2 pm.
Social testing was performed between 9 am and 5 pm. Fig. 2 describes the core and associated-
symptoms of ASD in human and how it can be evaluated in rodent ASD models. A schematic
diagram of the schedule of behavior and biochemical analysis for this project is shown in Fig. 3.
18
Fig. 1 Injection schedule of MIA dams. The day that the plug was observed is considered E0.5.
The litters are kept with their mother until weaning at PND21. Male and female offspring were
housed separately (2 to 4 mice per cage). Behavioral analysis commenced at 6 weeks.
19
Fig. 2 Translating human ASD behaviors to mouse model of ASD. The table lists some of the
mouse behavior tests that can be used for certain core and associated – ASD behaviors
manifested in humans (Crawley, 2004, Roullet and Crawley, 2011).
Deficit in social interaction and communication
Ritualistic Repetitive Behaviour
Human ASD behaviors Mouse Behavioral Tests
Gaze aversion eye blink test
Deficit in social -emotional reciprocity 3-chamber paradigm Deficit in developing and maintaining relationships tube test
Lack of initiation during social interaction
Language delay ultrasonic vocalization
Motor Stereotypies grooming marble burying test
Insistence on sameness reversal of a position habi t in an appetitive T -maze task reversal of a position habit in the Morris water maze
Hyperactivity motor activity test
Intellectual disability contextual and cued fear conditioning Operant learning tasks Acquisition of T -maze tasks
Seizures audiogenic seizure test
Sensory hyper/hyposensitivity prepulse inhibition Tactile startle
Sleep dysfunction observation of home cage sleep patterns
Increased anxiety elevated plus maze light-dark exploration open field test
Co
re B
eh
avio
rs o
f A
SD
Asso
cia
ted
Beh
avio
rs o
f A
SD
20
Fig. 3 The schedule of behavioral and biochemical analysis of MIA offspring.
3.2 Motor Activity
Motor activity was measured using the automated VersaMax animal activity monitoring system
(AccuScan Instruments, Inc., US) as previously described (Pacey et al., 2011). The animals were
tested at 6 weeks of age. The detection system consisted of a 42 x 42 x 30 cm Plexiglas box
placed inside the activity monitor that emitted 16 x 16 x 16 laser beams, each separated by 2.5
cm. The system measured the animal’s activity by tracking the number of beams that was
broken. Two smaller Plexiglas boxes (21 x 21 x 21 cm) were placed at diagonal corners inside
the larger Plexiglas box for activity measurement of each individual mouse. All animals were
habituated under dim lighting (the room was evenly lit by three 14 W incandescent light fixtures)
for at least one hour prior to testing. Before each test, the smaller Plexiglas box was wiped
thoroughly with Virox (0.4% hydrogen peroxide), followed by water. Thereafter, the animal was
gently placed in the center of the smaller Plexiglas box and a lid was immediately placed on top
to prevent the animal from escaping. The test was performed under dim lighting and the data
generated by the monitor was tabulated every 5 minutes for an hour. The following parameters
were measured: distance travelled, horizontal movement time, vertical activity, and vertical
movement time.
3.3 Social Interaction and Social Preference Test
The modified three chamber paradigm protocol was based on Ramsey et al., 2011 with some
modifications. The entire test was conducted under dim lighting (approximately 40 lux). A
21
digital camera was mounted on the ceiling approximately one meter above the social apparatus to
track the locomotion of the animal. Rather than physically separating the chambers, we virtually
defined two identical circular zones (diameter: 19.8 cm) in a white Plexiglas box (61.7 x 40.8 x
23 cm) using Viewer2 software (BIOBSERVE GmbH, NJ). These two zones were parallel from
each other and equidistant from the edge of the box (7.1 cm lengthwise, 10.5 cm widthwise). An
empty white wire cage (top diameter 7.5 cm, bottom diameter 10 cm, height 10 cm) was placed
at the center of each zone, covered by a white circular disk (diameter: 10 cm) and held down by a
standard 400 mL glass beaker. The beaker prevented the test animals from climbing on top of
the wire cages. Two removable, white cardboard dividers (40 x 23 cm) were inserted into the
middle of the box, shielding both zones. The activity of the animal in each zone was recorded
every 2 minutes for 10 minutes.
The stimulus animals (wild-type C57BL/6, age and sex matched with test animal) were always
kept at the opposite corner from the test animal to ensure its novelty. Stimulus animals were
habituated for at least 30 minutes in their respective home cage under dim lighting as previously
described. Each animal was then trained inside a clean cylindrical wire cage placed inside the
white Plexiglas box. A maximum of four stimulus animals were trained at the same time for two
15-minute sessions. The stimulus animals were used for subsequent social testing if no persistent
aggressive behavior was observed (e.g. climbing and/or biting of the cage).
All social testing was conducted at 7 to 8 weeks of age. Test animals were habituated for at least
one hour in their home cages under dim lighting as previously described. Before each test, all
apparatus were wiped vigorously with Virox, followed by water. To begin, the test animal was
gently placed between the dividers. Habituation commenced when the dividers were
simultaneously lifted, exposing the animal to the two zones, each with an empty wire cage held
down by a beaker as previously described. After the animal was allowed to habituate for 10 min,
it was then isolated between the two dividers again, shielding it from the two zones. If side
preference was present (i.e. the difference in time spent between the two zones was more than 30
seconds), the animal was returned to home cage and tested the next day. Otherwise, one stimulus
animal (of same sex and similar age) was placed in one of the wire cages while a novel object
was placed in the other cage. The designation of the zones was determined at random. The
novel object used was a green Biobag tied into a knot placed on top of a roll of green tape
(diameter: 6 cm). The dividers were then simultaneously lifted and the animal was allowed to
22
explore for 10 minutes. Social preference index for the social interaction test was calculated by
the difference in time spent between the two zones (social zone minus non-social zone) divided
by the sum of time spent in both zones. At the end of the social interaction test, the animal was
again isolated between the dividers to shield it from both zones. Another novel stimulus mouse
(of same sex and similar age) was placed in the non-social zone, replacing the novel object. This
now becomes the non-familiar zone. The dividers were then lifted simultaneously and the
animal was allowed to explore for 10 minutes. Social preference index for social preference test
was calculated by the difference of time spent between the two zones (non-familiar zone minus
familiar zone) divided by the sum of time spent in both zones. At the end of the experiment, the
test and stimulus animals were returned to their respective cages. A T-test was performed
between the social zone and non-social zone or familiar zone and non-familiar zone for each
treatment group separately to determine if there was a significant preference for one zone over
the other (Ehninger et al., 2012, Lipina et al., 2013).
3.4 Marble Burying Test
Marble burying test is a test for repetitive/compulsive behavior and was conducted at 10 weeks
of age as previously described (Thomas et al., 2009). Clean cages (29.5 x 17.5 x 12.5 cm) were
filled up to 5 cm with corn cob bedding. Twenty identical navy marbles (diameter: 1.2 cm) were
placed in a 4 x 5 rectangular matrix occupying 2/3 of the cage. The animals were first habituated
in the biosafety cabinet class A/B for at least 30 minutes with the blower on as background noise.
After habituation, the test mouse was gently placed into 1/3 of the cage without of the marbles
and a lid was immediately placed over the cage. Individual test animals were allowed to explore
the cage for 30 minutes after which they were returned to the home cages. The marbles were
cleaned with Virox and rinsed with water between each test. A marble was considered buried
when more than 50% of its surface area was covered by the bedding.
3.5 Grooming
Self-grooming behavior entails licking and scratching of any body parts of the mouse (Kalueff
and Tuohimaa, 2005). The total time spent grooming (in seconds) was recorded manually based
on previously recorded videos of the social preference test (first 5 minutes of exploration period).
The experimenter was blinded to the treatment group of the test animal.
23
3.6 Western Blot
Each cerebellum was dissected, frozen on dry ice, and stored at -80°C. Once one set of
cerebellar samples (i.e. saline, LPS1X, LPS 2X or saline, Poly IC 1X, Poly IC 2X; 2-3 sample
for each treatment group) have been accumulated, the tissues were homogenized in tandem on
the same day in lysis buffer (50 mM Tris-HCl, 1% SDS, 1X protease inhibitor cocktail (PIC,
Sigma-Aldrich, St. Louis, MO) pH 7.4). Protein concentrations were measured using BCA assay
kit (Sigma-Aldrich).
Equal amounts of total protein were loaded in each lane and electrophoretically separated on
12% polyacrylamide gels (one sample set per gel). The amount of total protein loaded (4 – 40
μg) depended on the abundance of the target protein in the cerebellum. The protein was then
transferred to a nitrocellulose membrane using the Bio-Rad Trans-Blot semi-dry transfer system
and blocked in wash buffer (0.01 M Tris, 0.015 M NaCl, 0.05% Tween-20, pH 7.6) with 5%
skim milk at room temperature for 30 minutes. The membrane was then washed 3 x 15 minutes
with wash buffer and incubated with primary antibodies (made in wash buffer) overnight at 4°C.
The primary antibodies used were as follows: anti-GFAP (1: 500, clone N206A/8, NeuroMab),
rabbit anti-Iba-1 (1: 250, Wako), rat anti-MBP (1:3000, Millipore), and anti-glyceraldehyde 3-
phosphate-dehydrogenase (GAPDH) (1:20 000 to 1: 100 000 depending on the amount of protein
loaded, Sigma-Aldrich). The next day, the membranes were washed 3 x 15 minutes with wash
buffer and incubated for 2 hours at room temperature in secondary antibodies (made in wash
buffer with 3% skim milk). The horseradish peroxidase-conjugated secondary antibodies used
were as follows: goat anti-rabbit (1:2500, Jackson ImmunoResearch), goat anti-mouse (1:5000,
Jackson ImmunoResearch), and donkey anti-rat (1:2500, Jackson ImmunoResearch). This was
continued by 3 x 15-minute washes with wash buffer. Immunoreactivity was detected using
SuperSignal West Pico Chemiluminescence substrate (Fisher Scientific, Pittsburgh, PA) and
imaged using Alpha Innotech FluorChem® Chemiluminescent Imaging System. The integrated
density value per area of the target bands were quantified using AlphaEaseFC image analysis
software normalized to the loading control (i.e. GAPDH). Each LPS sample set consisted of
saline (n = 3), LPS 1X (n = 3) and LPS 2X (n = 2 or 3). The protein expression of LPS treated
groups were expressed as a percentage of saline treated groups. The values from different
sample sets were then averaged between different sample sets.
24
3.7 Statistical Analysis
Statistical analysis was conducted only for groups of mice with n ≥ 8 and analyzed separately for
each treatment (i.e. Poly IC or LPS), genotype (i.e. wild-type or Fmr1 knockout) and sex.
Statistical analysis was performed using GraphPad Prism 5. For studies with two sample groups,
a student’s t-test was performed with 95% confidence interval. For studies with three sample
groups, one way ANOVA was performed followed by Bonferroni post hoc test. Grubbs’ test
was used to eliminate outliers.
25
4 Results
4.1 The Effect of MIA on Body Weight
The weight of MIA offspring was measured on PND46 as an indicator of their overall health.
Prenatal LPS treatment did not affect the weight of wild-type female (F(2, 40) = 0.2763, p =
0.7600) or male mice (F(2, 36) = 2.72, p = 0.0793) (Fig. 4A). Similarly, prenatal Poly IC
treatment did not alter the weight of wild-type female (F(2, 32) = 1.31, p = 0.2850) or male
offspring (F(2, 23) = 0.80, p = 0.4627) (Fig. 4B).
Saline Fmr1 KO mice were heavier in comparison to WT mice of the same treatment. This was
significant for male (t(24) = 2.87, p < 0.01) but not female mice (t(32) = 0.80, p = 0.4314).
Treatment with Poly IC decreased the weight of female (F(2, 38) = 3.81, p < 0.05; Bonferroni
post hoc test Poly 1X p > 0.05, Poly 2X p > 0.05) and male Fmr1 KO mice (F(2, 29) = 8.29, p <
0.01; Bonferroni post hoc test Poly 1X p < 0.01, Poly 2X p < 0.05) (Fig. 4C).
4.2 The effect of MIA on Motor Activity
Locomotor and exploratory behavior of the MIA offspring was investigated by measuring
horizontal and vertical activity in an automated activity box. It should be noted that for all
treatment groups, male mice demonstrated higher vertical activity compared to female mice.
LPS treatment (i.e. both LPS 1X and 2X) did not affect the horizontal activity in the offspring in
terms of total distance travelled (female: F(2, 54) = 0.09, p = 0.9181; male: F(2, 59) = 1.14, p =
0.3268) and total horizontal movement time (female: F(2, 54) = 0.01, p = 0.9908; male: F(2, 59)
= 1.24, p = 0.2958) (Fig. 5A, B). In addition, it did not affect the total vertical activity (female:
F(2, 54) = 2.75, p = 0.0728; male: F(2, 59) = 0.44, p = 0.6467) or total vertical movement time
(female: F(2, 54) = 3.05, p = 0.06, male: F(2, 59) = 0.70, p = 0.5020) (Fig. 5C, D).
Single Poly IC prenatal injection significantly decreased the total distance travelled (F(2, 52) =
7.17, p < 0.01, Bonferroni post hoc test p < 0.01) and total horizontal movement time (F(2, 52) =
6.28, p < 0.01, Bonferroni post hoc test p < 0.01) in male but did not affect female offspring.
26
Fig. 4 Body weight of wild-type and Fmr1 knockout MIA offspring on PND46. LPS (A)
and Poly IC (B) treatment did not affect the weight of wild-type mice. Prenatal Poly IC injection
significantly decreased the body weight of male Fmr1 KO mice (C). Each column represents the
average ± S.E.M. One-way ANOVA was performed for each genotype and sex separately,
followed by Bonferroni’s post hoc analysis. Student’s t-test was performed for saline WT vs.
saline KO. Significant outliers were removed using the Grubbs’ test. Statistical significance is
not shown for groups with n < 8. * p < 0.05, ** p < 0.01.
27
Fig. 5 Motor activity of LPS wild-type offspring. LPS treatment did not affect the total
distance travelled (A), total horizontal movement time (B), total vertical activity (C) and total
vertical movement time (D) of the offspring. Each column represents the average ± S.E.M.
One-way ANOVA was performed for each sex separately, followed by Bonferroni’s post hoc
analysis.
In contrast, double Poly IC prenatal injections significantly increased the horizontal activity of
female offspring with regards to total distance travelled (F(2, 61) = 4.24, p < 0.05; Bonferroni
post hoc test p < 0.05) and total horizontal movement time (F(2, 61) = 3.91, p < 0.05; Bonferroni
post hoc test p < 0.05) but did not affect the male offspring (Fig. 6A, B). Both Poly IC 1X and
2X treatment did not affect the total vertical activity (female: F(2, 61) = 0.40, p = 0.6699; male:
F(2, 52) = 0.57, p = 0.5685) and total vertical movement time (female: F(2, 61) = 1.26, p =
0.2911, male: F(2, 52) = 0.40, p = 0.6735) of MIA offspring (Fig. 6C, D).
Saline Fmr1 KO mice were hyperactive compare to saline wild-type mice with regards to total
distance travelled (female: t(40) = 8.66, p < 0.0001; male: t(32) = 6.28, p < 0.0001), horizontal
movement time (female: t(40) = 7.49, p < 0.0001; male: t(32) = 5.39, p < 0.0001), vertical
28
activity (female: t(40) = 4.51, p < 0.0001; male: t(32) = 4.79, p < 0.0001), and vertical movement
time (female: t(40) = 5.10, p < 0.0001; male: t(32) = 5.00, p < 0.0001) (Fig. 7A – D).
In Fmr1 KO mice, Poly IC treatment (1X and 2X) did not further increase the total distance
travelled (female: F(2, 45) = 2.07, p = 0.1378; male: F(2, 35) = 0.18, p = 0.8371) or horizontal
movement time (female: F(2, 45) = 2.57, p = 0.0876; male: F(2, 35) = 0.47, p = 0.6318) (Fig. 7A,
B). In contrast, Poly 1X and 2X female Fmr1 KO mice showed significantly decreased total
vertical activity (F(2, 45) = 6.86, p < 0.01, Bonferroni post hoc test p < 0.05 for Poly 1X, p <
0.01 for Poly 2X) and vertical movement time (F (2, 45) = 8.61, p < 0.001, Bonferroni post hoc
test p < 0.01). A similar trend was observed for Poly 1X male but not Poly 2X male Fmr1 KO
mice for total vertical activity (F (2, 35) = 3.09, p = 0.0582) and vertical movement time (F (2,
35) = 4.46, p < 0.05, Bonferroni post hoc test p < 0.05 for Poly 1X, p > 0.05 for Poly 2X).
To summarize the motor activity results, while prenatal LPS injection into wild-type mice did not
affect the motor activity of the offspring, prenatal Poly IC injection caused sex and treatment-
dependent changes in the horizontal activity of wild-type offspring and also significantly
decreased the vertical activity of Fmr1 knockout mice.
4.3 The Effect of MIA on Social Behavior
Social impairment is one of the core symptoms of ASD and is often linked with deficits in
communication. In rodents, this behavior can be assessed using the 3-chamber paradigm
developed by Crawley’s group (Silverman et al., 2010). This test was divided into two stages.
In the social interaction test, sociability was measured by comparing the time the animal spent in
the zone with novel mouse vs. novel object. In the social preference test, the second stage,
sociability was measured by comparing the time the animal spent in the zone with the familiar
mouse vs. non-familiar mouse. In both tests, normal social behavior entailed preference for the
social and non-familiar zone over the non-social and familiar zone, respectively.
29
Fig. 6 Motor activity of Poly IC wild-type offspring. For female offspring, double Poly IC
injection significantly increased the total distance travelled (A) and total horizontal movement
time (B). For male offspring, single Poly IC injection significantly decreased these two
parameters. Poly IC treatment did not affect the total vertical activity (C) and total vertical
movement time (D) of MIA offspring. Each column represents the average ± S.E.M. One-way
ANOVA was performed for each sex separately, followed by Bonferroni’s post hoc analysis.
* p < 0.05. *** p < 0.001.
30
Fig. 7 Motor activity of Poly IC Fmr1 knockout offspring. Compared to saline WT animals,
saline Fmr1 KO mice were significantly more hyperactive with respect to all four parameters (A
– D). Poly IC treatment did not affect the total distance travelled (A) and total horizontal
movement time (B) of Fmr1 KO offspring. Poly IC treatment decreased the total vertical activity
(C) and total vertical movement (D) of Fmr1 KO offspring with the exception of Poly 2X male
mice. Each column represents the average ± S.E.M. One-way ANOVA was performed for each
sex separately, followed by Bonferroni’s post hoc analysis. Student’s t-test was performed for
saline wild-type vs. saline knockout for each sex separately. * p < 0.05. ** p < 0.01. **** p <
0.0001.
31
LPS treatment caused decreased sociability in female mice only (Fig. 8). In the social interaction
test, LPS 1X female offspring showed no statistically significant preference towards the social
zone (saline: t(32) = 4.00, p < 0.001; LPS 1X: t(20) = 1.77, p = 0.0914; LPS 2X: t(14) = 3.01, p
< 0.01). As well, female LPS mice showed a trend towards lower social interaction index
suggesting their preference for the social zone was not as strong as the respective control groups
(F(2,33) = 1.942, p = 0.1595). In contrast, LPS male offspring displayed normal social behavior
by preferring the social zone over the non-social zone (saline: t(30) = 3.86, p < 0.01; LPS 1X:
t(20) = 6.30, p < 0.0001; LPS 2X: t(24) = 5.70, p < 0.0001) (Fig. 8A). The social interaction
index for male LPS offspring was comparable to that of the controls (F(2, 37) = 1.40, p = 0.2586)
(Fig. 8C). For social preference test, LPS offspring displayed decreased sociability by showing
no preference for the non-familiar zone (saline: t(28) = 2.93, p < 0.01; LPS 1X: t(20) = 0.080, p
= 0.9369; LPS 2X: t(14) = 0.83, 0.4223) (Fig. 8B). LPS female offspring also showed a trend
towards decreased social preference index (F(2, 33) = 2.57, p = 0.0916) (Fig. 8D). Male saline
and LPS offspring showed no preference for the non-familiar zone (saline: t(28) = 1.34, p =
0.1900; LPS 1X: t(20) = 1.31, p = 0.2002; LPS 2X: t(24) = 1.89, p = 0.0709) (Fig. 8B). Male
LPS offspring also had comparable social preference index with saline control animals (F(2, 31)
= 0.0082, p = 0.8591) (Fig. 8D).
Poly IC wild-type offspring showed no deficits in sociability. For female offspring, both Poly
1X and Poly 2X offspring displayed normal sociability by spending more time in the social zone
(Poly 1X: t(18) = 1.89, p = 0.0745), Poly 2X: t(14) = 2.96, p < 0.05) (Fig. 9A). Their respective
social interaction index did not significantly differ from controls (F(2, 32) = 1.39, p = 0.2643)
(Fig. 9C). Although both Poly 1X and 2X female offspring did not show a significant preference
for the non-familiar zone (Poly 1X: t(18) = 0.83, p = 0.4198, Poly 2X: t(14) = 1.07, p = 0.3050),
their social preference index was comparable to the saline group (F(2,32) = 0.83, p = 0.4466). In
comparison, Poly 1X male offspring demonstrated normal social behavior by spending more
time in the social zone (t(14) = 3.26, p < 0.01) and non-familiar zone (t(10) = 2.53, p < 0.05).
They also did not significantly differ from controls with respect to social interaction index (F(2,
28) = 0.49, p = 0.6151) and social preference index (F(2, 26) = 1.41, p = 0.2615) (Fig. 9C, D).
Statistical tests were not performed for Poly 2X male wild-type offspring because the n-value
was less than 8.
32
Fig. 8 Social behavior of LPS treated wild-type MIA offspring evaluated by the modified 3-
chamber paradigm. For social interaction test, only LPS 1X female mice displayed deficits in
sociability by showing no preference for the social zone (A). This deficit was reflected by the
decreased social interaction index for this group, although not statistically significant (C). For
social preference test, deficits in sociability were observed for both LPS 1X and LPS 2X female
mice where they showed no preference for the non-familiar zone (B). This lack of preference
was observed for all male groups, including control. The social preference index was decreased
for female but not male mice, although not statistically significant (D). Time spent in each zone
was measured in seconds. Each column represents the average ± S.E.M. One-way ANOVA was
performed for each sex separately, followed by Bonferroni’s post hoc analysis. Using the
Grubbs’ test, one value that was furthest from the mean was omitted for each group, except for
those with n ≤ 8. * p < 0.05. ** p < 0.01. *** p < 0.001.
33
Fig. 9 Social behavior of Poly IC treated wild-type MIA offspring evaluated by the
modified 3-chamber paradigm. For social interaction test, only Poly 1X female mice displayed
deficits in sociability by showing no preference for the social zone (A). This deficit was not
reflected by the social interaction index (C). For social preference test, deficits in sociability
were observed for all groups except for female saline and male Poly 1X offspring where they
showed a significant preference for the non-familiar zone (B). The Poly IC treated groups did
not differ in social preference index (D). Time spent in each zone was measured in seconds.
Each column represents the average ± S.E.M. One-way ANOVA was performed for each sex
separately, followed by Bonferroni’s post hoc analysis. Using the Grubbs’ test, one value that
was furthest from the mean was omitted for each group, except for those with n ≤ 8. * p < 0.05.
** p < 0.01. *** p < 0.001.
34
Similar to wild-type saline treated mice, both female and male saline treated Fmr1 KO mice
demonstrated normal social behavior during social interaction test by spending significantly
more time in the social zone (female: t(16) = 4.33, p < 0.001; male: t(16) = 3.56, p < 0.01) (Fig.
10A). However, in the social preference test, both female and male saline treated Fmr1 KO mice
showed no significant preference for either the non-familiar zone or the familiar zone (female:
t(16) = 1.54, p = 0.1355; male: t(16) = 0.29, p = 0.7745) (Fig. 10B).
With regards to the effect of Poly IC on Fmr1 KO mice, decreases in sociability was only
observed in female Poly 1X group where it did not show a preference for the social zone during
social interaction test (t(26) = 1.85, p = 0.07449) (Fig. 10A). This result was also reflected by a
significantly lower social interaction index in Poly 1X female mice compared to control (F(2, 32)
= 3.78, p < 0.05, Bonferroni post hoc test p < 0.05) (Fig. 10C). In contrast, Poly IC treatment did
not impair the sociability of other Fmr1 KO groups during social interaction test (Poly 2X
female: t(22) = 2.98, p < 0.01; Poly 1X male: t(16) = 3.56, p < 0.01; Poly 2X male: t(22) = 5.99,
p < 0.001). Comparison of social interaction index revealed the same findings for these groups
with the exception of Poly 2X male offspring which showed a trend towards decreased index
(female: F(2, 32) = 3.78, p < 0.05, Bonferroni post hoc test p > 0.05 for Poly 2X; male: F(2, 26)
= 4.79, p < 0.05, Bonferroni post hoc test p > 0.05 for Poly 1X and Poly 2X). For social
preference test, none of the groups demonstrated a preference for either zone with the exception
of female Poly 2X and male Poly 1X Fmr1 KO offspring that showed a preference for the non-
familiar zone (female Poly 1X: t(26) = 1.62, p = 0.1167; female Poly 2X: t(22) = 3.93, p < 0.001;
male Poly IX: t(22) = 2.47, p < 0.05; male Poly 2X: t(14) = 1.64, p = 0.1121) (Fig. 10B). Similar
results were observed with respect to the social preference index where a trend towards more
preference for the non-familiar zone was found for Poly 2X Fmr1 KO female (F(2, 32) = 2.54, p
= 0.0950) and Poly 1X Fmr1 KO male mice (F(2, 26) = 1.72, p = 0.1168) (Fig. 10D).
35
Fig. 10 Social behavior of Poly IC treated Fmr1 knockout MIA offspring evaluated by the
modified 3-chamber paradigm. For social interaction test, all groups demonstrated normal
social behavior except for female Poly 1X mice where it showed no significant preference for the
social zone (A). The impairment in this group was reflected by the significant decrease in social
interaction index (C). For social preference test, only female Poly 2X and male Poly 1X mice
showed normal social behavior by preferring the non-familiar zone (B). This result was partially
reflected in the social preference index where Poly 2X female showed increased score, although
not statistically significant (D). Wild-type saline groups (grey) were included as a comparison.
Time spent in each zone was measured in seconds. Each column represents the average ±
S.E.M. One-way ANOVA was performed for each sex separately, followed by Bonferroni’s post
hoc analysis. Using the Grubbs’ test, one value that was furthest from the mean was omitted for
each group. * p < 0.05. ** p < 0.01. *** p < 0.001.
36
It should be noted that the average time spent in all four zones did not significantly differ
between controls and MIA offspring. Modest deficits were only evident through social
interaction and/or social preference index. Despite this, the deficits observed in social behavior
were limited to female MIA offspring only. Single prenatal LPS treatment caused a strong trend
towards deficits in both social interaction and preference test. This impairment was maintained
in the offspring from double injected dams (i.e. LPS 2X), particularly in the last 5 minutes of the
exploration period (see Appendix 1). In contrast, only female offspring from Poly 1X FXS dams
showed deficits in social interaction and the deficits were not preserved in female Poly 2X
offspring. Therefore, social impairments in MIA offspring were dependent on sex and genotype
of the offspring as well as the immunological memory of the dam (for Poly IC only).
4.4 The Effect of MIA on Repetitive Behavior Evaluated Using Marble Burying Test
Ritualistic and repetitive behavior is the other core symptom of ASD. This behavior can be
assessed in rodents using the marble burying test where increased repetitive behavior is
correlated with more marbles buried (Thomas et al., 2009). The response of MIA offspring was
sex-dependent for this test. For female wild-type mice, MIA offspring showed no difference
with regards to the number of marbles buried (LPS: F(2, 46) = 1.98, p = 0.1237; Poly IC: F(2,
49) = 2.18, p = 0.1237). In contrast, both LPS and Poly IC male wild-type offspring buried
significantly more marbles compared to controls (LPS: F(2, 44) = 8.66, p < 0.001; Bonferroni
post hoc test LPS 1X: p < 0.05, LPS 2X: p < 0.001; Poly IC: F (2, 37) = 5.55, p < 0.01;
Bonferroni post hoc test p < 0.05) (Fig. 11A, B). Saline treated male but not female Fmr1 KO
mice showed a trend towards increased repetitive behavior compared to saline WT mice (female:
t(38) = 0.6257, p = 0.5352; male: t(28) = 1.903, p = 0.0673). Poly IC treatment did not
exacerbate the repetitive behavior of Fmr1 KO mice (female: F (2, 45) = 2.95, p = 0.0628, male:
F (2, 30) = 0.13, p = 0.8746) (Fig. 11C).
To summarize, MIA wild-type offspring demonstrated increased repetitive behavior that was
found in male but not female mice. For Fmr1 knockout mice, marble burying was not affected
by prenatal Poly IC treatment.
37
Fig. 11 Repetitive behavior of wild-type and Fmr1 knockout MIA offspring evaluated by
the marble burying test. Male wild-type LPS (A) and Poly IC (B) treated MIA offspring
displayed increased repetitive behavior by burying significantly more marbles compared to
control. For female wild-type MIA mice, the number of marbles buried was not different in 1X
offspring and decreased in 2X offspring, although not statistically significant. Compared to
saline wild-type mice, male saline Fmr1 knockout mice showed a trend towards more marble
burying. This increase was not exacerbated by Poly IC treatment in Fmr1 knockout mice (C).
Each column represents the average ± S.E.M. One-way ANOVA was performed for each sex
separately, followed by Bonferroni’s post hoc analysis. Using the Grubbs’ test, one value that
was furthest from the mean was omitted for each group, except for those with n ≤ 8. * p < 0.05.
** p < 0.01. *** p < 0.001.
38
4.5 The Effect of MIA on Repetitive Behavior Evaluated by Grooming
Repetitive behavior was also evaluated by scoring the time the animal spent grooming during the
first five minutes of social preference test. Similar to marble burying test, LPS female offspring
showed a trend towards decreased repetitive behavior by spending less time grooming (F(2, 33)
= 2.47, p = 0.1005). No significant differences in grooming was found in LPS male offspring
(F(2, 30) = 0.50, p = 0.6142) (Fig. 12A). Interestingly, the lack of change in repetitive behavior
(via marble burying test) in female Poly IC offspring was mirrored in grooming results of wild-
type (F(2, 32) = 0.05, p = 0.9547) and FXS (F(2, 31) = 0.06, p = 0.9463) animals. A strong trend
towards increased repetitive behavior was observed in both Poly IC 1X wild-type (F(2, 25) =
3.23, p = 0.06) and FXS (F(2, 25) = 3.09, p = 0.06) offspring but not Poly IC 2X offspring (Fig.
12B, C).
4.6 The Effect of MIA on the Adult Cerebellum Evaluated by Western Blot
Quantitative western blotting was performed to investigate the expression of markers for
microglia (Iba-1), astrocytes (GFAP) and mature oligodendrocytes (MBP) in the adult
cerebellum. A representative blot for each marker is shown in Fig. 13A – C and the summary of
data and statistics is shown in Fig. 13D. Interestingly, both female and male LPS1X offspring
had approximately the same magnitude of changes in the expression of Iba-1 (~16% increase),
GFAP (~20% increase) and MBP (~10% decrease) compared to control, although not
statistically significant. For MBP, the 17 and 18.5 kDa bands were analyzed together because
they were not well separated on the gel.
39
Figure 12 Repetitive behavior of MIA wild-type and Fmr1 knockout MIA offspring
evaluated by grooming behavior. This parameter was recorded during the first 5 minutes of the
social preference test. For female, only LPS 2X offspring (A) showed a trend towards decreased
grooming time while other groups did not differ from control. For male, single Poly IC prenatal
injection increased grooming time in both wild-type (B) and Fmr1 knockout (C) offspring,
although not statistically significant. Double Poly IC prenatal treatment did not affect grooming
time in male offspring compared to control (B, C). Each column represents the average ± S.E.M.
One-way ANOVA was performed for each sex separately, followed by Bonferroni’s post hoc
analysis. Using the Grubbs’ test, one value that was furthest from the mean was omitted for each
group, except for those with n ≤ 8.
40
Fig. 13 Summary of the expression of different glial markers in the adult cerebellum of
LPS offspring quantified by western blot. Representative blots are shown in A – C. Ionized
calcium binding adaptor molecule 1 (Iba-1) (A), glial fibrillary acidic protein (GFAP) (B), and
myelin basic protein (MBP) (C) were used as markers for microglia, astrocytes, and
oligodendrocytes, respectively. Numerical values and statistics are tabulated in D. Each value
represents the average (expressed as a percentage of the respective saline controls) ± S.E.M.
One-way ANOVA was performed for each sex separately. Cerebellar tissue samples were
collected between PND70-90.
41
5 Discussion
5.1 The Effect of MIA on Body Weight
Sickness behaviors such as weight loss have been reported in animals injected with LPS
(Cloutier et al., 2012) or Poly IC (Cunningham et al., 2012). We postulated that the immune
response from dams injected with these agents will perturb the development of the fetal brain.
However, it is important that the immune responses were not severe enough to become
detrimental to the well-being of the offspring since autistic patients are typically healthy and
have normal body weight (Bolte et al., 2001, Curtin et al., 2005). Therefore, we decided to
assess the general health of the offspring by measuring their weight on PND46.
We found that prenatal LPS injections did not affect the weight of the wild-type offspring (Fig.
4A). Similar results were observed by other groups where they found normal body weight in
offspring from dams that received an acute LPS injection during early or late gestation (Golan et
al., 2005, Ling et al., 2009). It should be noted that chronic LPS injections to the dam decreased
the body weight of male offspring (PND21 to adulthood) (Bakos et al., 2004). In contrast, a high
dose (790 μg/kg) of LPS injection during early pregnancy (compared to 75 μg/kg used in this our
animal model) increased the body weight of adult offspring (Nilsson et al., 2001, Wei et al.,
2007). Therefore, the effects of LPS on offspring weight gain may be dependent on the
treatment regime of the dams. Our model used a low and acute dose of LPS which could explain
the lack of effect on the body weight of the litters.
Saline treated male Fmr1 KO offspring were significantly heavier compared to wild-type mice
(Fig. 4C). This sex difference in body weight was also observed at PND30 by Pacey et al. 2011.
In addition, Bhattacharya et al. 2012 reported that the weight gain was significantly increased
after 6 weeks in male mice which was approximately the age that we weighed our animals. This
weight gain mirrored the accelerated prepubescent growth in human Fragile X patients (Loesch
et al., 1995). Moreover, we found that prenatal Poly IC injection significantly decreased the
weight of male but not female Fragile X offspring (Fig. 4C). This decrease was absent in Poly
IC wild-type animals (Fig. 4B). Several groups with different treatment regimens reported that
Poly IC injection during pregnancy did not affect the weight of wild-type offspring from birth to
early adulthood (Yee et al., 2011, Malkova et al., 2012, Vorhees et al., 2012). However,
offspring from dams that demonstrated a weight loss following Poly IC injection showed reduced
42
weight at approximately three months of age (Bronson et al., 2011, Vorhee et al., 2012). It is
possible that because body weight was assessed at a single time point, we were unable to capture
the weight change later on in life. It is also possible that the genetic predisposition of Fmr1
knockout mice contributed to an earlier onset of weight loss caused by prenatal Poly IC
treatment. A longitudinal analysis of body weight would help us determine if this was the case.
5.2 The Effect of MIA on Motor activity
Motor activity was tested to evaluate locomotion and exploratory behavior in a novel
environment. Similar to published findings (Golan et al., 2005, Girard et al., 2009, Zager et al.,
2012), acute prenatal LPS treatment did not affect the horizontal or vertical motor activity of the
offspring (Fig. 5). The lack of change in locomotor activity could be due to the serotype of LPS
as well as the frequency and dose of LPS given prenatally. For example, Ling et al., 2009
showed that prenatal injection of a much higher dose of LPS (1 mg/kg) during early gestation
increased the locomotor activity of the offspring at PND90.
Several groups reported that offspring from Poly IC injected dams showed decreased total
distance travelled during open field test (Meyer et al., 2006, Smith et al. 2007, de Miranda et al.,
2010, Malkova, et al, 2012). Some groups also reported thigomotaxis behavior where the Poly
IC offspring spent less time in the center of the open field box (Ozawa et al., 2006, Smith et al.,
2007, Soumiya et al., 2011). Similar to these previous reports, we found that male Poly IC 1X
offspring travelled less distance compare to respective controls (Fig. 6). However, this was not
observed in the female Poly IC 1X offspring or male Poly IC 2X offspring. Rather, female Poly
2X offspring showed significantly increased horizontal activity compare to controls. Most
groups analyzed either only male offspring or a combination of male and female offspring. To
our knowledge, no published reports have studied female Poly IC offspring specifically. It is
possible that the effect of Poly IC on motor activity could be sex-dependent. For example, in the
valporic acid model of ASD, Kim et al., 2013 reported that locomotor activity was only affected
in male and not female offspring.
With regards to thigomotaxis behavior, we did not find that Poly IC animals preferred to occupy
the margin zone more than the center zone (data not shown). This could be because we tested
the animals in smaller activity boxes under dim lighting whereas other groups tested the animals
43
in larger activity boxes under bright lighting. The latter test conditions may have created a more
stressful environment for the animals, hence inducing thigomotaxis behavior.
In agreement with published literature, we found that both male and female Fmr1 KO mice
exhibited hyperactivity (Spencer et al, 2005, Pacey et al, 2011). Although prenatal Poly IC
treatment did not affect the horizontal activity of Fragile X mice, it did significantly decrease the
vertical movement of both female and male offspring with the exception of male Poly 2X FXS
mice (Fig. 7). Vertical activity can be considered as a form of exploratory behavior that is
associated with the animals’ arousal state and attention in a novel environment (Sadile et al.,
1995, Aspide et al., 1998, 2000). In fact, decreased vertical activity (i.e. rearing) has been
observed in other ASD models including Gabrb3 knockout mice (DeLorey et al., 2008) and wild-
type LPS MIA mice (Liu et al., 2004). Interestingly, Walker et al., 2007 reported significant
decreases in exploratory behavior in adult male rats that underwent developmental cerebellar
suction lesions or intracerebroventricular saporin injections specifically targeting the Purkinje
cells. The association between cerebellar pathologies and lack of exploratory behavior was
reflected in a human study as well. Pierce and Courchesne, 2001 showed that decreased
exploration in autistic patients, measured by the time spent investigating different novel objects
in a large room, was correlated with the extent of cerebellar hypoplasia of vermal lobules VI-VII.
Therefore, decreases in both horizontal and vertical activity could be considered as ASD-
associated phenotypes in mice, and the cerebellum might be a key brain region linked to these
behaviors.
5.3 The Effect of MIA on Social behavior
Impairments in social interaction and communication are one of the core symptoms of ASD.
Since the symptoms persist throughout life, we decided to investigate the social behavior of the
adult MIA offspring. First, prenatal infections caused moderate deficits in female offspring only
(Fig. 8, Fig. 10). All male MIA groups displayed normal social behavior, evident by
demonstrating preference of the social and non-familiar zone. As well, the male social
interaction and social preference index did not significantly differ from the respective controls.
Second, tandem injections of LPS (i.e. LPS 2X) maintained the deficits in social behavior
observed in the LPS 1X female offspring (Fig. 8). Lastly, whereas Poly IC had no effects in any
of the wild-type injected mice, Poly IC 1X treatment did induce deficits in female Fragile X
44
knockout animals, suggesting a possible genotype effect (Fig. 10). Unlike the LPS 2X female
offspring, tandem injections of Poly IC in FXS animals did not preserve the social deficits
observed in Poly 1X female offspring (Fig. 10).
Overall, the deficits observed in social behavior were modest since the average time the MIA
offspring spent interacting with the social or non-familiar zone did not significantly differ from
the respective saline controls. Deficits were only apparent through the social interaction and/or
preference index. Previous published findings have reported social impairments in both the LPS
(Golan et al., 2006, Kirsten et al., 2010, 2012) and Poly IC model (Shi et al., 2003, Smith et al.,
2007, Soumiya et al., 2011, Malkova et al., 2012). Possible reasons why we did not see such
strong impairments are discussed below.
Kirsten et al. 2010 investigated sociability in rats prenatally treated with LPS by subjecting the
offspring to novel free moving stimulus animal in a cage and manually scoring their play
behavior such as sniffing and mounting. They found that they were only able to detect deficits in
social interaction in adult rats when they isolated the rats (i.e. one rat per cage) for a week before
testing. This was done to increase the rats’ motivation to engage in social interaction with the
novel stimulus rat. We housed our animals in a group setting without social isolation prior to
testing. Thus lower social motivation in the test animals may explain why we were unable to
detect stronger deficits in the MIA model.
Contrary to our findings, Kirsten et al, 2010, 2012 found male-specific deficits in social
interaction in the rat LPS model. They found significant reductions in the male offspring’s
frequency to crawl under/over and mount on the stimulus rat. These two parameters are more
aggressive behaviors that are usually more common in male rodents. Female mice are generally
more docile. For example, male rats had a mounting frequency of 4 (Kirsten et al., 2010) during
the 30 minutes of exploration whereas female only had a frequency of 2.5 (Kirsten et al., 2012).
Since the frequency was already reduced in female control rats, deficits may be harder to detect
in the LPS offspring. Furthermore, they found no difference in less aggressive social behaviors
(i.e. sniffing and following stimulus rat) in either male or female adult LPS rats compared to
controls. Our test condition was less aggressive in comparison because the stimulus mice were
constrained in wire cups and were not in direct contact with the test mouse. Therefore, the lack
45
of social interaction in female rats observed by Kirsten et al. 2012 does not conflict with the
social deficits we observed using the modified 3-chamber paradigm.
In the Poly IC model, deficits in sociability were previously reported in three different strains
including C57/B6 (Smith et al., 2007, Malkova et al., 2012), ddY (Soumiya et al., 2011), and
BALB (Shi et al. 2003) mice using the 3-chamber paradigm assay. It should be noted that all
findings were reported by Patterson’s group with the exception of Soumiya et al., 2011. The
impairments were present in either only male mice or a combination of both female and male
mice. To our knowledge, no sex differences in social interaction analyses have been specifically
investigated. It should be emphasized that we used a modified version of the 3-chamber
paradigm. Rather than chambers, we confined the area of social interaction to zones which was
much smaller in size. Therefore, interaction with either the stimulus mouse or novel object was
only recorded when the test mouse was in close proximity. This may be a more stringent
measure because in the typical 3-chamber paradigm, time is recorded after the animal enters the
chamber, even though they may not be actually interacting with the stimulus.
Although we were unable to detect a difference in sociability in the Poly IC wild-type animals,
we did find significant decreases in social interaction index in the FXS Poly IC 1X female
offspring (Fig. 10). This suggested that the lack of FMRP in the FXS animals may have
rendered them more vulnerable in the presence of maternal Poly IC injection. Ehninger et al.,
2012 performed a similar study using Tsc2 halpodeficient mice, a genetic model of tuberous
sclerosis which also has high incidences of ASD (Moss et al., 2009). Similar to our results, they
found that deficits in social interaction were only observed in Tsc2 halpodeficient mice (not
wild-type mice) with mid-gestational viral infection using Poly IC. Tsc2 is able to from a
complex with Tsc1 to negatively regulate mammalian target of rapamycin complex 1
(mTORC1). Interestingly, cytokines such as TNF-α are able to inhibit the formation of Tsc1 and
Tsc2 complex via activation of Akt (also known as protein kinase B) (Lee et al, 2007).
Therefore, changes in levels of cytokines plus dampened function of Tsc2 may elevate mTORC1
signaling. The mTORC1 signaling pathway plays an important role in the regulation of the
innate and adaptive immune system (Weichhart et al., 2008, Thomson et al. 2009). For example,
mTORC1 is able to activate signal transducer and activator of transcription 3 (STAT3) which is
essential for the differentiation of Th17 cells (Stepkowski et al., 2008). Interestingly, Sharma et
al., 2010 found that mTOR phosphorylation and activity were elevated in the hippocampus of
46
juvenile Fmr1 knockout mice. This suggests that deficits observed in Poly IC 1X FXS offspring
may also be linked to disruptions in the mTOR pathway.
In summary, deficits in social interaction were limited to female offspring only. Prenatal
bacterial (LPS) not viral infection (Poly IC) to naïve dams caused modest social impairments in
the female offspring. Furthermore, this deficit was preserved in offspring from LPS dams with
immunological memory (i.e. LPS 2X wild-type). Lastly, prenatal viral infection in the Fmr1 KO
mice caused deficits in social behavior that was not observed in Poly IC treated wild-type
animals, suggestive of possible gene x environment interaction.
5.4 The Effect of MIA on Ritualistic and Repetitive Behavior
Ritualistic and repetitive behavior is the other core symptom of ASD. According to DSM-V, it
can be manifested through stereotyped motor movements, ritualistic behavior (e.g. insistence on
sameness), highly restricted interests, and hyper or hypo-reactivity to sensory input. These
behaviors can be evaluated in animals via different assays. The most established tests to assess
stereotyped motor movements are the marble burying assay and self-grooming behavior (Roullet
and Crawley, 2011). Rodents that exhibit an autistic-like phenotype will bury more marbles and
spend more time grooming.
Our results from the marble burying tests and grooming analyses showed a striking sex
difference in the MIA mice. In female offspring, LPS treatment caused a trend towards
decreased marble burying and grooming (Fig. 11A, 12A) whereas Poly IC treatment caused no
changes in either parameter (Fig. 11B, 12B). In contrast, both male LPS and Poly IC offspring
showed increased repetitive behavior by burying significantly more marbles compared to
controls (Fig. 11A, B). The male-specific increases in repetitive behavior were also observed in
male Poly 1X wild-type offspring where they spent more time grooming (Fig. 12A).
Other groups have reported increased repetitive behavior using the MIA model. Malkova et al.,
2012 found significant increases in marble burying and grooming in male Poly IC offspring. As
well, Kirsten et al., 2012 found that male LPS offspring with an additional LPS exposure had
increased reluctance to switch arms in the T-maze spontaneous alternation test. Although this
test was used to evaluate learning and spatial memory, they suggested that it may also reflect
increased insistence on sameness, which is a form of repetitive behavior. Increased time spent
47
grooming has been observed in several different ASD mouse models. These include MIA Poly
IC model (Malkova et al., 2012) as well as genetic models of ASD including BTBR T+tf/J mice
(McFarlane et al., 2008, Pearson et al., 2011) and Shank 3 mutant mice (Peca et al., 2011).
However, all of the groups mentioned only used male mice to assess the repetitive behavior.
We saw a male-specific trend towards increased marble burying in saline FXS mice compared to
saline wild-type mice. Similarly, Bhattacharya et al., 2012 reported significant increases in the
number of marbles buried in FXS animals compared to WT animals. However, it should be
noted that three papers published by Paylor’s group found no significant difference between
wild-type and FXS animals (Veeraragavan et al., 2011, Henderson et al., 2012, Thomas et al.,
2012). Both groups used mice with the same genetic background as our animals (C57/B6). One
explanation for this disparity could be that the bedding Paylor’s group used (i.e. SANI-CHIP)
was much easier to bury with than the corn cob bedding used in our study. In fact, the wild-type
animals in their study buried almost 3 times more marbles than in our study. Prenatal Poly IC
treatment to Fmr1 knockout mice caused a trend towards increased grooming time (similar to
WT animals) but did not exacerbate the number of marbles buried. The lack of change in marble
burying is likely due to the ceiling effect where the increases in control Fmr1 KO mice narrowed
the window that Poly IC treatment could exert. In other words, the Fmr1 KO mice may have
already reached the maximum number of marbles that they are able to bury.
In addition to repetitive behavior, increases in self-grooming and marble burying may be
associated with increased anxiety, although there are some caveats. Increases in self-grooming
occur in both low-stress and high-stress situations. In low-stress environments, mice tend to
groom for longer period of time and follow normal progression of grooming activity. In contrast,
during high-stress conditions, mice generally have more bursts of short grooming activity with
abnormal progression, in addition to numerous incomplete and interrupted bouts (Kalueff et al.,
2004). Therefore, more comprehensive analyses of grooming behavior should to be conducted to
identify whether increases in grooming was related to increased anxiety. In terms of marble
burying test, several groups have reported that administration of various anxiolytic (alprazolam,
diazepam, chlordiazepoxide) or antidepressant (paroxetine, fluoxetine, citalopram) drugs
significantly decreased the number of marbles buried (Njung’e and Handley, 1991, Nicolas et al.,
2006, Gomez et al., 2011). However, administration of anxiogenic agents (pramipexole, methyl-
β-carboline-3-carboxylate, yohimbine, meta-chlorophenyl) not only did not increase the number
48
of marbles buried but significantly decreased burying behavior (Njung’e and Handley, 1991,
Nicolas et al., 2006, Gomez et al., 2011). Furthermore, Thomas et al., 2009 found that marble
burying behavior did not correlate with anxiety in open field test and light-dark exploration.
This suggests that marble burying may be more suitable as a predicative test to screen the effects
of anxiolytic drugs rather than a direct test for anxiety. Therefore, self-grooming and marble
burying test may, at best, only partially reflect anxiety in rodents. More specific tests such as
elevated plus maze and open field test may be more suitable for evaluating anxious behavior.
Our study was the first to demonstrate the sex difference in stereotyped repetitive behavior in
MIA animal models. The strong sex-bias observed in marble burying assay and grooming is
reflected in human ASD patients. In fact, several studies demonstrated that males have higher
repetitive and/or restricted interests compared to females (Hattier, 2011, Sipes et al., 2011,
Mandy et al., 2012, Werling et al, 2013). However, I should note that there are also published
studies that found no gender differences in young ASD patients in terms of repetitive behavior
(Worley et al., 2011 (children and adolescent), Andersson et al., 2013 (pre-school children with
suspected of ASD)). In addition, the lack of change in motor stereotypies in females does not
exclude the fact that they may still demonstrate other forms of repetitive behavior such as
restricted interest and ritualistic behavior.
Both animal and human studies suggest that increased repetitive behavior may be linked to
cerebellar pathologies. In rodents, increases in repetitive behavior were reported in mice with
mutations restricted to Purkinje cell degeneration (i.e. Tsc1 mutant mice, Tsai et al., 2012) or
depletion of Purkinje cells during development (i.e. lurcher mice, Martin et al, 2010, Dickson et
al., 2010). In humans, Rojas et al., 2006 reported significant negative partial correlations
between grey matter volume in specific regions of the cerebellum (i.e. left cerebellar lobule VI
and right cerebellar Crus 1) and severity of repetitive and stereotype behaviors in 24 male ASD
patients. Similarly, in a study with 14 ASD patients, Pierce and Courchesne, 2001 found that the
area of cerebellar vermis lobules VI – VII was significantly negatively correlated with the rates
of stereotyped behavior. These studies suggest that increased repetitive behavior may be linked
to cerebellar pathologies.
A summary chart of all behavioral findings for wild-type and Fmr1 knockout MIA offspring can
be found in Appendix 2.
49
5.5 The Effect of MIA on the Adult Cerebellum
We wanted to investigate the pathology of the cerebellum because this brain structure has been
found to be consistently implicated in autism spectrum disorder. Our goal was to investigate
whether the pathologies observed in human ASD patients are present in our MIA mouse model.
First, we wanted to investigate if the presence of neuroinflammation persisted until adulthood.
We expected to see increases in microglia and/or astrocyte expression because this was observed
in human post-mortem tissues of ASD patients (Vargas et al, 2005). As well, several groups
have shown increases in the expression of microglia in certain brain regions of the LPS model
(Larouche et al, 2005, Roumier et al., 2008, Girard et al, 2010) and Poly IC model (Jackel et al.,
2011). Ling et al., 2009 reported that a single LPS injection of during early gestation increased
activated microglia in substantia nigra of adult offspring at 4, 14, and 17 months. Our results
thus far also show possible increases in microglia in the adult cerebellum (Fig. 13). However, it
was difficult to detect microglia marker (Iba-1) on western blots because the expression was very
low in whole cerebellar samples and thus required large amount of protein to be loaded.
Therefore, for future experiments, immunohistochemistry may be a better method for evaluating
the density of microglia in this particular brain structure.
Increases in the expression of astrocyte marker (GFAP) were reported in several brain regions of
the LPS model (Cai et al., 2000, Paintlia et al., 2004, 2008). Our results thus far showed
approximately a 20% increase in GFAP expression in the cerebellum of both male and female
LPS 1X groups, although not statistically significant. One possible reason for this could be
because we used tissue homogenates of the entire cerebellum. If GFAP expression was
increased in only certain regions of the cerebellum, analysis with the entire tissue may mask the
changes in a specific region(s).
Apart from the changes in microglia and astrocytes, several groups also reported decreases in
expression of MBP, a marker for mature oligodendrocytes. This was reported for various ages in
several brain regions including periventricular white matter at PND7 (Kumral et al., 2007,
Yesilirmak et al., 2007), corpus callosum at PND9 – 30 (Paintlia et al., 2004, 2008), and external
and internal capsule at PND7 (Rousset et al., 2006, 2008). Decreases in MBP were also found in
the hippocampal CA1 and CA3 region of Poly IC treated mice at PND14 (Makinodan et al.,
2008). Thus far, no group has reported MBP changes in the cerebellum. Our results showed
50
decreases in MBP in both LPS 1X and LPS 2X female and male offspring, although not
statistically significant. It may be beneficial to confirm this with another myelin marker such as
2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNPase) or proteolipid protein (PLP) to determine
if the decrease is real and reproducible.
To summarize, western blotting results were suggestive of potential signs of neuroinflammation
and white matter changes in LPS offspring. However, the n-values for these experiments were
too low to reach a firm conclusion.
5.6 Potential Biological Mechanisms for Sex-Dependent Behavioral Changes
In the marble burying test, we found that prenatal immune activation caused significant male-
specific increases in repetitive behavior. This suggests that the immune response maybe more
severe in male rather than female mice. Some explanations for this sex difference in immune
response are as follows. First of all, the placenta, derived from maternal and fetal origin may
contribute to the sex differences during prenatal programming (Rossant and Cross, 2001). In the
placenta of mothers with mild asthma, the mRNA expression of various cytokines (TNFα, IL-1β,
IL-6, IL-8, and IL5) was increased for female but not male fetuses (Scott et al., 2009). The
production of these cytokines can affect the hypothalamus-pituitary-adrenal axis which is
involved in the stress pathway activation and immune system. Cytokines such as IL-1β, which is
up-regulated after LPS injection, can promote the release of corticotropin releasing hormone and
adrenocorticotropic hormone from the hypothalamus and pituitary, respectively (Irwin and
Miller, 2007). In turn, glucocorticoids production, triggered by adrenocorticotropic hormone,
can readily pass through the placenta and participate in fetal brain development. In fact, Cui et
al., 2011 found LPS injection during mid or late-gestation increased the plasma
adrenocorticotropic hormone and corticosterone (a main glucocorticoid) levels in the dam. One
of the most important functions of glucocorticoids is its regulation of placental glucose
transporters. Exogenous glucocorticoid administration led to the reduction of glucose transporter
1 placental expression. However, this reduction was maintained in males but not in females later
in gestation (O’Connell et al., 2011).
Another explanation for the sex differences could be contributed to genetic factors. In contrast to
males who have only one X chromosome, females have two X chromosomes where the alleles
51
on one or the other are inactive. However, approximately 15% of X-linked genes escape this
inactivation to some degree (Carrel et al., 2005). There are many immune related genes on the
X-chromosome. These include immune-associated receptors (e.g. IL receptors), immune-
response related proteins (e.g. ELK1 – involved in B-cell development), and transcriptional and
translational control effectors (e.g. NF-kB repressing factor) (Fish, 2008). This could explain the
differences in female and male fetuses’ response to immune activation. In fact, 65 genes were
found to be changed in the placentae of mothers with chronic asthma. Interestingly, only 6 genes
were altered in male placentae compared to the 59 genes that were altered in female placentae.
The genes investigated are involved in a variety of functions including immune response, cellular
growth, cell-cell signaling, and gene expression (Osei-Kumah et al., 2011). Depending on the
function of the gene, dramatic changes in gene expression could be a protective mechanism for
female fetuses that is lacking in the male fetuses.
52
6 Conclusions
The main goal of this thesis was to evaluate the validity of the MIA mouse model for autism
spectrum disorder by investigating the core and associated-ASD behaviors and adult cerebellar
pathologies. We found that prenatal bacterial infection via LPS to naïve dams induced both core
symptoms of ASD in wild-type offspring (i.e. LPS 1X). In addition, the deficits in social
interaction and increases in repetitive behavior were preserved in LPS 2X offspring. In
comparison, prenatal viral infection (Poly IC) in wild-type offspring was only able to increase
the repetitive behavior.
Interestingly, although social interaction was not impaired in Poly 1X and 2X wild-type mice,
prenatal Poly IC injection to naïve dams impaired the social behavior in Fmr1 knockout mice. In
fact, prenatal Poly IC treatment caused other phenotypes in the Fmr1 knockout mice that were
absent in the Poly IC wild-type mice. These included significant reductions in body weight and
rearing. Genotype-specific impairments in Poly IC Fmr1 knockout mice suggested that the lack
of FMRP protein may have rendered the mice more vulnerable to prenatal viral infection.
Lastly, prenatal bacterial and viral infection caused striking sex-dependent phenotypes that were
absent in saline control mice. Specifically, we found that deficits in social interaction were
female-specific whereas increases in repetitive behavior were male-specific. In addition, western
blots performed on the adult cerebellum were suggestive of possible increases and decreases in
the expression of astrocytes and mature oligodendrocytes, respectively. To summarize, we found
that the ASD-related behavioral phenotypes observed in the MIA model were dependent on the
sex and genotype of the offspring, type of prenatal infection, and/or immunological memory of
the dam.
53
7 Future Directions
Although we investigated the core symptoms of ASD in this study, it would be beneficial to see
if these core phenotypes can be replicated using other behavioral assays. One of the most
interesting effects observed based on the MIA models was the strong male-specific increases in
motor stereotypies. It would be interesting to investigate if the sex difference is preserved in
other forms of repetitive behavior. For example, insistence on sameness can be evaluated using
the reversal learning tasks to assess the likelihood of the animal to change their established
habits. Restricted interest is another form of repetitive behavior that can be measured by
counting the frequency of nose-pokes into holes baited with a stimulus. We also found female-
specific modest deficits in social interaction. This may be improved by subjecting the animals to
isolation prior to testing to increase their motivation to engage in social interaction. As well, we
can look at the progression of the social behavior across different time intervals. Other assays
can be used to assess other forms of social behavior such as social dominance using the tube test.
We did not specifically investigate communication in the MIA model, although it is linked with
social behavior. This can be evaluated using ultrasonic vocalization and measuring pup retrieval
time by the dams. Associated-ASD behaviors such as anxiety should also be investigated.
Grooming is a type of repetitive behavior that may be linked with increased anxiety (Kalueff et
al., 2004, Thomas et al., 2009). Grooming in high stress situations involves “frequent bursts of
rapid short grooming activity with abnormal progression and frequent incomplete and interrupted
bouts” (Fentress, 1977). It may be worthwhile to perform more in-depth analyses of grooming
behavior to identify if excessive grooming observed in MIA offspring is caused by the high
stress social environment during the social preference test. If grooming was increased due to
stress, it may be of interest to use more specific assays that are catered to anxiety such as open
field test, light/dark box, and elevated plus maze test. Animals that are more anxious will spend
less time in the open arm, brightly lit area of the box, and center of the open field.
Molecularly, we can further our investigation on the presence of neuroinflammation in the adult
cerebellum of the MIA model. Our results thus far with the LSP model were suggestive of
possible changes in the expression of microglia, astrocyte and oligodendrocyte markers.
Additional analyses using immunohistochemistry would be helpful to identify the particular
region that may be affected. Furthermore, the number and size of Purkinje cells could be
54
examined to determine whether the reductions observed in ASD patients are present in the MIA
model. Lastly, since Fmr1 knockout mice showed more severe phenotypes after prenatal Poly IC
treatment compared to control, it would be interesting to elucidate the possible pathway(s)
involved. Further investigation of the mTOR pathway may be a fruitful endeavor since it is
misregulated (over-active) in Fragile X syndrome and is involved in the modulation of both the
innate and adaptive immune responses.
55
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Appendices
Appendix 1 Comparison between computer and manual scoring of social interaction of
LPS treated wild-type MIA female offspring evaluated by the modified 3-chamber
paradigm. The computer (A) and manual (B) analysis of social interaction test results were
comparable. The similarity between the two methods was also reflected by the social interaction
index (D, E). When the analysis was performed separately for each half of the exploration
period, both LPS 1X and LPS 2X offspring showed deficits in sociability in the last 5 minutes
(C). This result was reflected by the social interaction index (F). Each column represents the
average ± S.E.M. One-way ANOVA was performed, followed by Bonferroni’s post hoc
analysis. Using the Grubbs’ test, one value that was furthest from the mean was omitted for each
group, except for those with n ≤ 8. * p < 0.05. ** p < 0.01. *** p < 0.001. **** p < 0.0001.
83
Appendix 2 Summary of behavioral findings in wild-type MIA offspring and Fmr1
knockout MIA offspring. The solid red line represents the behavioral outcome of control saline
WT (A) and saline FXS (B) mice. The light grey areas represent a trend and the dark grey areas
represent statistical significance of at least p < 0.05.
Appendix 3. Summary of literature research regarding Poly IC MIA rodent model. All findings are of offspring from Poly IC injected dams in comparison to its respective control. N/A: not applicable. 84
Author (Year) Target Disease Species (Strain)
Dose (route)
Time of Injection
Behavioral Findings Cytokine Findings Neuroinflammation-related Findings
Arrode-Bruses et al. (2012)
not specified mouse (C57/B6)
20 mg/kg (i.p.)
E16 N/A pro-inflammatory cytokines (IL-1β, IL-13),
chemokines (MCP-1, MIP-1α, IP-10, MIG) and CSF VEGF in fetal brain (6 or
24 hr after injection)
N/A
Bronson et al. (2011)
Schizophrenia rat (Sprague-Dawley)
8 mg/kg (i.p.)
E 14 offspring showed bimodal weight change; male offspring from Poly IC dams with weight loss had decreased MK801-stimulated (P56) and
amphetamine-stimulated (P90) locomotion
compared to offspring from Poly IC dams with weight gain and vehicle dams; weight loss of the
Poly IC dam increased with increasing maternal age and more frequent
social isolation
N/A N/A
Dickerson et al. (2012)
schizophrenia rat (Sprague-Dawley)
4.0 mg/kg (iv)
E15 theta frequency coherence between the
prefrontal cortex and hippocampus was
significantly reduced in the MIA group during baseline resting-state
recordings (adult)
N/A N/A
Appendix 3. Summary of literature research regarding Poly IC MIA rodent model. All findings are of offspring from Poly IC injected dams in comparison to its respective control. N/A: not applicable. 85
Author (Year) Target Disease Species (Strain)
Dose (route)
Time of Injection
Behavioral Findings Cytokine Findings Neuroinflammation-related Findings
Forrest et al. (2012)
not specified rat (Wistar)
10 mg/kg (i.p.)
E14, E16, E18
N/A ↑ MCP-1 in serum of Poly IC dams (5 hr after
injection)
N/A
Gilmore et al. (2005)
not specified rat (Sprague-Dawley)
10 or 20 mg/kg (i.p.)
E16 N/A 20 mg/kg: TNFα (plasma and placenta, no
change in fetal brain) 10 mg/kg: ↓ TNFα whole
brain (P1)
N/A
Han et al. (2011)
not specified rat (Sprague-Dawley)
0.5 mg/kg (i.p.)
daily from E15 to E18
no difference in spatial learning (Morris water maze); poly IC offspring
showed impaired reversal learning; Poly IC offspring had higher TNFα in serum
(> p35)
N/A N/A
Hsiao and Patterson
(2011)
neurodegenerative disease
mouse (C57/B6)
20 mg/kg (i.p.)
E12.5 ↓ in PPI, ↑ % freezing, ↓ entries into the center of
the open field (P42)
N/A N/A
Juckel et al. (2011)
Schizophrenia mice (BALB/c)
20 mg/kg (i.p.)
E9 N/A N/A in Iba1-positive cells in hippocampus and
striatum, not frontal and occipital cortex
Koga et al. (2009)
not specified mouse (C57/B6)
4.5 mg/kg (i.p.)
E16.5 N/A IL6, MCP-1, IL-12p40, RANTES, KC, MIP-1β in the placenta (2 hr, 4 hr
after injection)
↑ NF-ĸB activation in the placenta (2hr after
injection)
Li et al. (2009) Schizophrenia mouse (C57/B6)
5 mg/kg (iv)
E9 or E17 E9: ↓ in PPI (P91); E17: no change in PPI (P91)
N/A N/A
Appendix 3. Summary of literature research regarding Poly IC MIA rodent model. All findings are of offspring from Poly IC injected dams in comparison to its respective control. N/A: not applicable. 86
Author (Year) Target Disease Species (Strain)
Dose (route)
Time of Injection
Behavioral Findings Cytokine Findings Neuroinflammation-related Findings
Makinodan et al. (2008)
Schizophrenia mouse (C57/B6)
60 mg/kg (i.p.)
E9.5 ↓ PPI (P63) N/A ↓ mRNA for MBP in hippocampus, ↓ myelin thickness in hippocampal
CA1; ↓ MBP immunostaining in
hippocampal CA1 and CA3 (P14)
Malkova et al. (2012)
Autism mouse (C57/B6,
male)
5 mg/kg (i.p.)
E10.5, E12.5, E14.5
↓ number of USV to social stimuli (P10, 8-9
weeks); ↓ PPI (6 weeks); ↓ total distance travelled in open field (7 weeks); ↓
social interaction (10 weeks); deficit in female
urine-induced scent marking (11 weeks); ↑
marble burying (12 weeks); self-grooming (13 weeks); no change in olfactory sensitivity (13--
14 weeks)
N/A N/A
Mandal et al. (2010, 2011)
neurodegenerative disease
mouse (C57/B6)
20 mg/kg (i.p.)
E12 N/A increase in IL6 in the amniotic fluid is partly
contributed by the fetus
Lymphocytes from offspring of Poly (I:C)
injected immune dams showed preferential
ability to develop into Th 17 cells compared to PBS-injected control and Poly
(I:C) injected immunologically naïve
dams
Appendix 3. Summary of literature research regarding Poly IC MIA rodent model. All findings are of offspring from Poly IC injected dams in comparison to its respective control. N/A: not applicable. 87
Author (Year) Target Disease Species (Strain)
Dose (route)
Time of Injection
Behavioral Findings Cytokine Findings Neuroinflammation-related Findings
Meyer et al. (2006)
Schizophrenia mouse (C57/B6)
5 mg/kg (iv)
E9 no change in latent inhibition (P35-45), disruption in latent inhibition (P95-135)
N/A N/A
Meyer et al. (2006)
Schizophrenia, neurodegenerative
diseases
mouse (C57/B6)
5 mg/kg (iv)
E9 or E17 Injection at E9: ↓ PPI (P98-P112); ↓ in open-field exploration (P98-112); Injection at E17:
preservative behaviour (reversal learning of left-
right discrimination) (male only P98-112); ↓
working memory in water maze (P98-P112)
E9 and E17: IL-1β, IL6, IL-10, TNFα in brain (3 hr after injection); ↑ IL-6, IL10 in brain (6 hr after
injection)
N/A
Meyer et al. (2008)
Schizophrenia mouse (C57/B6)
5 mg/kg (iv)
E9 ↑ AMPH- and/or MK801-induced locomotion (P35,
P100-110)
N/A N/A
Meyer et al. (2008)
Schizophrenia mouse (C57/B6)
5 mg/kg (iv)
E9 ↓ PPI (P90-P120); deficit in PPI was improved by
periadolescent treatment of clozapine and
fluoxetine
N/A N/A
Miranda et al. (2010)
neurodegenerative disease
mouse (C57/B6)
5 mg/kg (i.p.)
E16 or E15-17
E16: ↓ in open-field locomotor activity (P8);
↓ in PPI at 4 dB and 8 dB (P8)
N/A N/A
Ozawa et al. (2006)
Schizophrenia mouse (Balb/c)
5 mg/kg (i.p.)
E12-E17 ↓ PPI, ↑ thigmotaxis, ↓ novel object recognition;
↑ MAP-induced locomotion (P63-P70)
N/A N/A
Appendix 3. Summary of literature research regarding Poly IC MIA rodent model. All findings are of offspring from Poly IC injected dams in comparison to its respective control. N/A: not applicable. 88
Author (Year) Target Disease Species (Strain)
Dose (route)
Time of Injection
Behavioral Findings Cytokine Findings Neuroinflammation-related Findings
Shi et al. (2003)
neurodegenerative disease
mouse (Balb/c)
20 mg/kg (i.p.)
E9.5 ↓ PPI (P42-P56) N/A N/A
Shi et al. (2009)
neurodegenerative disease
mouse (C57/B6)
20 mg/kg (i.p.)
E12.5 N/A N/A ↓ cerebellar Purkinje cells (P11)
Smith et al. (2007)
neurodegenerative disease
mouse (C57/B6)
20 mg/kg (i.p.)
E12.5 ↓ PPI, ↓ LI, ↓ exploration in open field,
↓ social interaction (adult, age unspecified)
N/A N/A
Soumiya et al. (2011)
neurodegenerative disease
mouse (ddY)
20 mg/kg (i.p.)
E9.5 ↑ anxiety (i.e. reluctance to enter the center
portion of the well-lit open field) and ↓ in
social interaction (i.e. showed no preference for
social chamber)
N/A N/A
Appendix 3. Summary of literature research regarding Poly IC MIA rodent model. All findings are of offspring from Poly IC injected dams in comparison to its respective control. N/A: not applicable. 89
Author (Year) Target Disease Species (Strain)
Dose (route)
Time of Injection
Behavioral Findings Cytokine Findings Neuroinflammation-related Findings
Vorhees et al. (2012)
Schizophrenia rat (Harlan
Sprague-Dawley)
8 mg/kg (i.p.)
E14 ↓ in the rate of route-based learning; ↓
prepulse inhibition of acoustic startle in females (not males), exaggerated hyperactivity in response to (+)-amphetamine, and attenuated hyperactivity in response to MK-801; no change in elevated zero maze, open-field
activity, object burying, light-dark test, straight
channel swimming, Morris water maze spatial
acquisition, reversal, or shift navigation or spatial
working or reference memory, or conditioned
contextual or cued fear or latent inhibition (P65-87)
N/A N/A
Wolff and Bilkey (2008)
Schizophrenia rat (Sprague-Dawley)
4 mg/kg (iv)
E15 ↓ PPI (P56) N/A N/A
Wolff et al. (2011)
schizophrenia rat (Sprague-Dawley)
4.0 mg/kg (iv)
E15 ↓ time spent exploring novel object; ↑ reversal
learning; more rapid within trial habituation in
the novel context recognition (adult)
N/A N/A
Appendix 3. Summary of literature research regarding Poly IC MIA rodent model. All findings are of offspring from Poly IC injected dams in comparison to its respective control. N/A: not applicable. 90
Author (Year) Target Disease Species (Strain)
Dose (route)
Time of Injection
Behavioral Findings Cytokine Findings Neuroinflammation-related Findings
Yee et al. (2011)
not specified rat (Sprague-Dawley)
4.0 mg/kg (tail vein)
E15 ↓ prepulse inhibition; ↓ litter size; ↑ pup weight
(PND2),↑ anxiety in elevated maze
no difference in IL6 serum level (P69), TNFα and IL-1β was under the limit of
detection
N/A
Yee et al. (2012)
not specified rat (Sprague-Dawley)
4 mg/kg (tail vein)
E15 ↑ total number of 22-kHz ultrasonic vocalization
during fear conditioning compared to control
(>P71); No difference in habituation, fear
conditioning, or fear testing in terms of rearing, grooming,
defecation or US induced jumps;
N/A N/A
Zuckerman et al. (2003,
2005)
Schizophrenia rat (Wistar)
4 mg/kg (iv)
E15 or E17 ↓ LI, ↑ reversal learning of left-right
discrimination, ↑ AMPH- and MK801-induced
locomotion (P90)
N/A N/A
Appendix 4. Summary of literature research regarding LPS MIA rodent model. All findings are of offspring from LPS injected dams in comparison to its respective control. N/A: not applicable. 91
Author (Year)
Target Disease Species (Strain)
Serotype of LPS
Dose (route)
Time of injection
Behavioural findings Cytokine Findings Neuroinflammation-related Findings
Ashdown et al.
(2006)
Schizophrenia rat 0111:B4 50 μg/kg (i.p.)
E18 N/A ↑ IL-1β, IL-6, TNFα in maternal plasma (2–4 hr after injection); ↑
IL-1β, IL-6, TNFα in placenta (2–8 hr after injection); ↑ IL-1β in
fetal plasma (4 hr after injection)
N/A
Bakos et al. (2004)
chronic inflammation
rat (Wistar)
0111:B4 (L3012)
20, 20, 40, 40, 80 μg/kg
(N/A)
E15-19 ↓ locomotor activity in elevated plus maze,
↑ slips in the beam walking test (not
significant) (female offspring only)
N/A N/A
Borrell et al. (2002)
Schizophrenia rat (Wistar)
026:B6 (L-3755)
1 mg/kg (sc) alternate days for whole
pregnancy
↓ PPI (P70, P100, P200)
N/A N/A
Cai et al. (2000)
PVL, associated with cerebral palsy
rat (Sprague-Dawley)
055:B5 500 μg/kg (i.p.)
E18-19 N/A N/A ↑ GFAP in hippocampus,
thalamus, hypothalamus, frontal
cortex but not cerebellum (P8), ↓
MBP (P8), no change microglia (OX-42 and
tomato lectin)
Appendix 4. Summary of literature research regarding LPS MIA rodent model. All findings are of offspring from LPS injected dams in comparison to its respective control. N/A: not applicable. 92
Author (Year)
Target Disease Species (Strain)
Serotype of LPS
Dose (route)
Time of injection
Behavioural findings Cytokine Findings Neuroinflammation-related Findings
Chang et al. (2011)
not specified mouse (CD1)
N/A 100 μg/kg (intrauterine)
E15/20 N/A ↑ IL6 expression in myometrium and
placenta which was attenuated by N-acetylcysteine in
myometrium; in fetal brain, ↑ IL6, TNFα,
and IL-1β
N/A
Coyle et al. (2009)
hypozincaemia mouse (C57/B6)
0111:B4 300 μg/kg (sc)
E8 ↑ exploration of familiar vs. novel
object (reversed by administering
zinc)(P84); no change spatial learning or
memory (cross-maze escape task) (P65,
P79)
N/A N/A
Dowling et al. (2012)
not specified rat (Spraque-Dawley)
055:B5 1 mg/kg (i.p.) E 19 in the placenta: CCL2, IL6, and TNFα,
this was reversed with the administration of mangesium sulfate
N/A N/A
Fortier et al. (2004)
Schizophrenia rat (Sprague-
Dawley Male)
0128:B12 (L-2755)
50 μg/kg (i.p.)
E18-19 ↓ PPI, ↑ AMPH-induced locomotion, enhanced acoustic
startle responses (P70)
N/A N/A
Fortier et al. (2007)
Schizophrenia rat (Sprague-
Dawley Male)
0111:B4 (L-2630)
50 or 100 or 50 μg/kg
(i.p.)
E10-11 or E15-16 or
E18-19
↓ PPI E15-16 and E18-19 (P70), no change in
PPI E10-11 (P70)
N/A N/A
Appendix 4. Summary of literature research regarding LPS MIA rodent model. All findings are of offspring from LPS injected dams in comparison to its respective control. N/A: not applicable. 93
Author (Year)
Target Disease Species (Strain)
Serotype of LPS
Dose (route)
Time of injection
Behavioural findings Cytokine Findings Neuroinflammation-related Findings
Gayle et al. (2004)
not specified rat 100 μg/kg (i.p.)
E18 N/A mRNA for IL-1β, IL6, TNFα in placenta
(1–24 hr after injection); ↑ IL-1β,
IL6, TNFα in amniotic fluid (1–12 hr after
injection); no change in these cytokine
levels was observed in the fetal brain (1-24 hr
after injection)
N/A
Gilmore et al. (2005)
Neurodevelopmental disorders
rat (Sprague-Dawley)
055:B5 100 μg/kg (i.p.)
E14-16 N/A ↓ TNFα in frontal cortex (P6)
N/A
Girard et al. (2009)
cerebral palsy rat (Lewis)
0127:B8 200 μg/kg (i.p.)
twice daily from
E17 to birth
↓ in weight before P3, no change in total
distance travelled or number of crossing
events in motor activity test; ↓ latency to fall off rotarod (P30,
P35, P40)
N/A N/A
Girard et al. (2010)
Neurodevelopmental disorders
rat (Lewis)
0127:B8 200 μg/kg (i.p.)
twice daily from
E18-20
motor coordination deficit by rotarod
(P30, P35, P40)
↑ IL-1β, TNFα, IL6, IL-1Ra in placenta
↑ CD68 (labyrinth) and proliferating
microglia (brain white matter) (P9)
Appendix 4. Summary of literature research regarding LPS MIA rodent model. All findings are of offspring from LPS injected dams in comparison to its respective control. N/A: not applicable. 94
Author (Year)
Target Disease Species (Strain)
Serotype of LPS
Dose (route)
Time of injection
Behavioural findings Cytokine Findings Neuroinflammation-related Findings
Golan et al. (2005)
Neurodevelopmental disorders
mouse (C57/B6)
N/A 120 μg/kg (i.p.)
E17 no change in exploratory behaviour,
sensory gain, and motor function;↓
associative learning in cued version of water maze, ↑ novel object
recognition,
↑ IL6 in fetal brain (3 hr after injection)
N/A
Golan et al. (2006)
Neurodevelopmental disorders
mouse (C57/B6)
N/A 120 μg/kg (i.p.)
E17 ↑ exploration in open field (P240, P600); ↑ anxiety (elevated plus
maze), ↓ social activity, ↓
aggressiveness towards cagemates (P240); age is a risk
factor for later neurodegenerative
diseases, not systemic maternal
inflammation
N/A N/A
Haesaert and Ornoy
(1986)
Neurodevelopmental disorders
mouse (-) 026:B6 (Difco Co.)
2.5 μg/kg (-) E10 , E12 ↑ audiogenic seizures (P28)
N/A N/A
Kumral et al. (2007); Yesilirmak
et al. (2007)
PVL, associated with cerebral palsy
rat (Sprague-Dawley)
055:B5 500 μg/kg (i.p.)
E18-19 N/A ↑ IL-1β, IL-6, TNFα in whole brain (P7)
↓ MBP immunostaining in
periventricular white matter (P7)
Appendix 4. Summary of literature research regarding LPS MIA rodent model. All findings are of offspring from LPS injected dams in comparison to its respective control. N/A: not applicable. 95
Author (Year)
Target Disease Species (Strain)
Serotype of LPS
Dose (route)
Time of injection
Behavioural findings Cytokine Findings Neuroinflammation-related Findings
Lante et al (2007, 2008)
Neurodevelopmental disorders
rat (Sprague-Dawley)
055:B5 500 μg/kg (i.p.)
E19 ↓ spatial learning in water maze (P28)
N/A N/A
Larouche et al.
(2005); Girard et al. (2010)
Hypoxia/Ischemia rat (Lewis)
0127:B8 200 μg/kg (i.p.)
twice daily from
E17 to birth
N/A N/A ↑ proliferating and total microglia in forebrain white
matter (P9)
Ling et al. (2002, 2004,
2006); Zhu et al.
(2007)
Parkinson's disease rat (Sprague-Dawley)
026:B6 (L8274)
1 mg/kg (i.p.) E10.5 N/A ↑ TNFα in striatum (P21, P120, P210,
P510) and SN (P210)
N/A
Ling et al. (2006, 2009)
Parkinson's disease rat (Sprague-Dawley)
026:B6 (L8274)
1 mg/kg (i.p.) E10.5 N/A N/A ↑ activated microglia in SN basally (P120,
P210, P420, P510) and in response to
postnatal intranigral LPS (P210-P294)
Ling et al. (2009)
Parkinson's disease rat (Sprague-Dawley)
026:B6 (L8274)
1 mg/kg (i.p.) E10.5 to E11
↑ locomotor activity (P90); ↓ locomotor
activity (P480)
N/A N/A
Liu et al. (2004)
Ethanol consumption
rat (Sprague-Dawley)
026:B6 (L3773)
1 mg/kg (sc) alternate days for whole
pregnancy
↑ ethanol intake and preference, ↓ rearing
(P100-P130)
N/A N/A
Appendix 4. Summary of literature research regarding LPS MIA rodent model. All findings are of offspring from LPS injected dams in comparison to its respective control. N/A: not applicable. 96
Author (Year)
Target Disease Species (Strain)
Serotype of LPS
Dose (route)
Time of injection
Behavioural findings Cytokine Findings Neuroinflammation-related Findings
Liverman et al.
(2006)
cerebral palsy mouse (C57/B6)
0127:B8 50 μg (i.p.) E18 N/A N/A ↑ expresion of MCP-1, IL6, IL-1β, VEGF; YB-
1, needin; ↓ expression of
semaphorin 5b and groucho (RT-PCR was performed on fetal
brain removed 0.5-6 hr after injection)
Ning et al. (2008)
not specified mouse 0127:B8 500 μg/kg (i.p.)
E17 N/A ↑ TNFα in fetal brain, liver and
amniotic fluid (1.5 hr after injection); no
change in mRNA for TNFα in fetal brain
(1.5 hr after injection)
N/A
Paintlia et al. (2004,
2008)
PVL, associated with cerebral palsy
rat (Sprague-Dawley)
055:B5 700 or 1000 μg/kg (i.p.)
E18 N/A ↑ mRNA for IL-1β (1-48 hr after injection), TNFα (1-24 hr after
injection) in fetal brain
↓ OPCs and immature oligodendrocytes in corpus callosum and
lateral ventricles with ↓ mRNA for MBP and PLP in brain; ↓ MBP,
↑ GFAP immunostaining in
corpus callosum and/or cingulum (P9,
P16, P23, P30)
Romero et al. (2007,
2010)
Neurodevelopmental disorders
rat (Wistar)
026:B6 (L-3755)
2 mg/kg (sc) daily for whole
pregnancy
↓ PPI (P35, P70, P170, P180, P400)
↑ in IL2, IL6, TNFα in adult rat serum
N/A
Appendix 4. Summary of literature research regarding LPS MIA rodent model. All findings are of offspring from LPS injected dams in comparison to its respective control. N/A: not applicable. 97
Author (Year)
Target Disease Species (Strain)
Serotype of LPS
Dose (route)
Time of injection
Behavioural findings Cytokine Findings Neuroinflammation-related Findings
Roumier et al.
(2008)
Neurodevelopmental disorders
mouse (C57/B6)
055:B5 120 μg/kg (i.p.)
E15 N/A N/A ↑ microglial density in hippocampus (P0)
Rousset et al. (2006,
2008)
PVL, associated with cerebral palsy
rat (Wistar)
055:B5 300 or 400 μg/kg (i.p.)
E19-20 N/A ↑ mRNA for IL-1β (P1), ↓ mRNA for
TNFα (P7) in whole brain
↓ MBP immunostaining in
external and internal capsule (P7); ↑
ibotenate-induced microglial activation
and astrogliosis in various regions (P9)
Salminen et al.
(2008)
not specified mouse (C57/B6)
0111:B4 1 mg/kg (i.p.) E16 or 17 N/A ↑ TNFα, MCP-1, and IL6 in the maternal serum and amniotic fluid; ↑ TNFα and
MCP-1 in fetal serum (1, 3, or 8 hr after
injection)
N/A
Urakubo et al.
(2001)
Schizophrenia rat (Sprague-Dawley)
055:B5 0.5 mg/kg (i.p.)
E16 N/A 0.5 mg/kg: in IL-1β, IL6, TNFα in placenta
and in IL6 in amniotic fluid (2 or 8 hr after injection) 2.5 mg/kg: in IL6, TNFα
in placenta and TNFα in amniotic fluid, ↓TNFα in fetal brain (2 hr after injection)
N/A
Appendix 4. Summary of literature research regarding LPS MIA rodent model. All findings are of offspring from LPS injected dams in comparison to its respective control. N/A: not applicable. 98
Author (Year)
Target Disease Species (Strain)
Serotype of LPS
Dose (route)
Time of injection
Behavioural findings Cytokine Findings Neuroinflammation-related Findings
Wang et al. (2010)
Neurodevelopmental disorders
mouse (CD1)
0127:B8 8 μg/kg (i.p.) daily from E8-15
↓ learning and retention in radical
arm maze (P200 female, P400 male); ↑ anxiety in open field,
impaired beam walking (P200 female);
↑ weight burrowed (P70 male, P600
female); ↓ weight hoarded (P200
female)
N/A N/A
Wijkstra et al. (1991)
Sexual behaviour rat (Wistar)
N/A 0.2 or 2 μg/kg (iv)
E18 ↑ latency to initiate sexual behaviour, ↓
intromission by males (P91)
N/A N/A
Zager et al. (2012)
not specified mouse (Swiss)
0127:B8 120 μg/kg (i.p.)
E17 no change in total locomotion and mean velocity. After acute
amphetamine treatment, total
locomotion and mean velocity compare to control and baseline
(P70-90)
N/A N/A
