Early neurodevelopmental consequences of maternal immune activation
in male and female Wistar rats
Joanna C Neill1, Michelle E Edye1, Ben Grayson1, Katie N Murray2, Joanna Oladipo1, Patrick C
McHugh3, Michael K Harte1, Irene Knuesel4 , Eric Prinssen4 ,
(1) Division of Pharmacy & Optometry, Faculty of Medicine, Biology and Health, University of Manchester, Oxford Road, Manchester, M13 9PT, UK.
(2) Departments of Neurology and Neuroscience, Yale University, New Haven, Connecticut, 06510, USA.
(3) Centre for Biomarker Research, School of Pharmacy, University of Huddersfield, Huddersfield, HD1 3DH, UK.
(4) Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel, 124 Grenzacherstrasse, Basel, CH 4070, Switzerland.
Introduction
Model Optimisation cont.
Results – qPCR & IHC
Repeated poly I:C does not induce further elevation
of plasma IL-6 in non-pregnant Wistar rats
mIA alters gene expression in male offspring
hippocampus at PD21
IL-6
G
2500
***
**
Poly
I:C-induced
maternal
immune
activation (mIA) in rodents is emerging as a
key model for NDDs.
Methods
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5mg
10mg
1500
$
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2.5mg
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38
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$
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$
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*
1000
Vehicle
2.5mg
5mg
10mg
*
*
37
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Day 1
Day 2
Day 3
Day 4
3h
Ba 6
se h
lin
e
6h
Day 5
3h
Day 5
Ba
se
lin
e
Day 3
3h
B
as 6h
e li
ne
36
0
3h
Ba 6
se h
lin
e
500
Day 1
We aim to establish a rat mIA model and
investigate early neurobiological changes in
male and female offspring.
Vehicle
2000
MIA induced a significant reduction in synaptic protein shank3 (A) and
increases in the microglia marker olfml3 (E) and astrocyte marker
GFAP (F) in male offspring.
Temperature
H
3h
B
as 6h
e li
ne
Epidemiology and genetics suggest a link
between prenatal inflammatory challenge
and neurodevelopmental disorders (NDDs)
such as schizophrenia. However, the
mechanisms involved remain unresolved.
IL-1β
Iba1
Merge
Figure 2. Cytokine response is not maintained with repeated
dosing, unlike temperature increases (G-H). For H, $ = 2.5 mg
vs saline; # = 5 mg vs saline; * = 10 mg vs saline.
Results – Maternal response
mIA induces a robust acute immune response in
the pregnant dam
Plasma IL-6 was elevated at 3h post poly I:C (A) but this was not
maintained when the dam was culled at PD21 (C). No change to
maternal weight over gestation (B) or significant effect on litter size
for either the GD21 (D) or PD21 (E) study was observed.
Figure 5. 10 mg/kg poly I:C on GD15 induces changes in
offspring hippocampal gene expression. Data points
represent mean litter value from 2 offspring per sex per litter;
n=4-5 litters. Data presented as mean ± SEM.; *P<0.05,
**P<0.01. Shank3, SH3 and multiple ankyrin repeat domains
3; Sema3a, semaphorin 3a; Mfsd2a, major facilitator super
family domain containing 2a; MBP, myelin basic protein;
Olfml3, olfactomedin like 3; GFAP, glial fibrillary acidic
protein.
mIA increases activated microglia in hippocampus
of male offspring at PD21
Pilot data in male offspring shows that 10 mg/kg poly I:C induces an
increase in amoeboid microglia, indicative of an active state.
Figure 3. 10 mg/kg poly I:C or saline injected i.p. on GD15.
n=5-10 per treatment; data are presented as mean ± SEM;
****P<0.0001.
Acute 10 mg/kg poly I:C induces a robust immune
response in non-pregnant Wistar rats
mIA induces a reduction in placenta weight but no
change to offspring body weight or IL-6
A
B
GD21 Body weight
6
GD21 Placenta weight
*
0.8
4
2
Saline
Poly I:C
0
Placenta weight (g)
Model Optimisation
Results – Morphological changes
Body weight (g)
qPCR: mRNA expression was normalised to SDHA
housekeeping gene and expressed as fold change from a
calibrator using 2-ΔΔCt.
Stats: Statistical analysis on non pregnant rats was
performed using Kruskal-Wallis test followed by Dunn’s
multiple comparison test (Figure 1) or a repeated measures
two-way ANOVA followed by Bonferroni’s multiple-comparison
test (Figure 2). To prevent litter effects skewing data, the
mean value per litter is presented (Figure 4-6) Student’s t-test
was performed or a Mann-Whitney test was used when
parametric analysis was not applicable.
0.6
Conclusions
0.4
0.2
Saline
Poly I:C
0.0
Male
Female
Figure 6. Microglia in 30µm hippocampal slices from male
offspring were stained using Iba1. Number of microglia
classed as resting (A), intermediate (B) or amoeboid (C) are
shown. Counting areas were randomly placed over the
hippocampus. Background grey data points represent
individual male offspring from n=3 dams; foreground black
data points represent litter means. Data are presented as
means ± SEM of multiple counting frames for 6 slices per
brain. Counting was performed by an experimenter blinded to
treatment. *P<0.05 for t test between offspring means.
Male
Female
Poly I:C at 10mg/kg i.p. on GD15 induced
early neurodevelopmental changes in the
offspring of Wistar rats without long term
effects on the dam.
mIA resulted in altered hippocampal gene
expression linked to glia and synaptic
function in male offspring on PD21.
Figure 1. Wistar rats produce the most robust elevation in
plasma IL-6 in response to acute 10 mg/kg poly I:C (A-C).
*P<0.05, **P<0.01, ***P<0.001. For D-F, $ = 5 mg vs saline; #
= 10 mg vs saline; * = 15 mg vs saline.
Figure 4. 10 mg/kg poly I:C induces a reduction in placenta
weight in female offspring but no change to offspring body
weight at GD21 or PD21. No effect on plasma IL-6
concentration was observed at PD21. Data presented as
mean ± SEM; n=5 (litter means); *P<0.05.
This model provides an in depth
longitudinal evaluation of mIA. This
approach may provide new targets for
drug discovery leading to improved
therapeutic strategies for NDDs.
[email protected]
Poster Template from www.manchester.ac.uk/photographics
mIA induced microglial activation in the
hippocampus of male offspring at PD21.