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Paper submitted within the scope of the Master’s Thesis
Master of Industrial Sciences
GROUP T – Leuven Engineering College – 2012-2013
Group T does not guarantee error-free content of this paper.
Steenackers Wim1
1Master student biochemistry, GROUP T – Leuven Engineering College, Vesaliusstraat 13, 3000 Leuven
De Herdt Eric2
2Unit Life, GROUP T – Leuven Engineering College, Vesaliusstraat 13, 3000 Leuven, eric.de.herdt@groept.be
,
De Boever Patrick3, Bos Inge3,4 , Int Panis Luc³
3Unit MRG, VITO, Vlasmeer 7, 2400 Mol
4Department Human Physiology and Sports Medicine, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Elsene
ABSTRACT
Air pollution exposure as well as physical activity was found to have different effects on the brain. At first, air
pollution is believed to evoke neuroinflammation which might induce neurodegeneration. Second, physical
activity benefits brain function by supporting neuroprotective effects and neurogenesis. Previously, we found
exercise induced up regulation of Brain-Derived Neurotrophic Factor (BDNF). BDNF is a neurotrophine
important for neurogenesis and neuroprotection. The purpose of this study was to examine the acute and short-
term effects of both exercise and air pollution on the brain, using gene expression analysis and whether these
effects translate to the blood. The gene expression of inflammatory markers (NFE2L2, IL1α, IL1β, IL6, TNFα,
NOS1, NOS2, NOS3 and COX2), neurotrophic growth factors (VEGFa, IGF1) and synaptic proteins (SYN1,
SYP) were analyzed in two different animal studies. At first, twenty C57BL6 mice were divided into four groups
of five mice and placed into the Craeybeckxtunnel with or without filter cap (Antwerp) for five days. Two
control groups were placed outside the tunnel. Brain samples of olfactory bulb and hippocampus were collected
twenty-four hours after the exposure to analyze changes in gene expression of inflammatory genes and brain
plasticity related genes. Second, twenty-four male, albino, Wistar rats were allocated into four groups to
investigate the effects of exposure to ultrafine particles (UFP) during a single bout of physical activity. The
animals were exposed for 90 minutes to one of the following regimes: Ambient air/Rest, Ambient air/Exercise,
UFP/Rest and UFP/Exercise. Analysis of changes in gene expression of inflammatory and brain plasticity related
genes was performed by qPCR. Previous analysis revealed an increased expression of inflammatory genes
(COX2, NOS2, NOS3, NFE2L2) in the hippocampus of the mice due to air pollution exposure. In this study, an
increased NOS1 expression was found due to air pollution in the mice hippocampus. The previously decreased
expression of BDNF did not reflect to the other brain plasticity related genes (VEGFa and IGF1). The rat study
previously revealed an up regulation of Brain-Derived Neurotrophic Factor (BDNF). In this sequel, synapsin 1
(SYN1) follows the same pattern as BDNF but without a significant increase due to exercise and with significant
decrease due to UFP, regardless of physical activity. The gene expression pattern in the brain did not seem to
translate to the blood. In addition to the previous findings demonstrating a negative effect of UFP exposure on
BDNF gene expression, this study shows a negative effect of UFP exposure on the gene expression of SYN1 and
VEGFa. This finding suggests that a short exposure to air pollution decreases neurotrophic support and synaptic
plasticity.
Keywords
Particulate matter (PM), brain-derived neurotrophic factor (BDNF), inflammation, hippocampus, olfactory bulb,
prefrontal cortex, blood, brain plasticity related genes.
NEUROINFLAMMATION INDUCED BY AIR POLLUTION: GENE
EXPRESSION ANALYSIS IN LABORATORY ANIMALS
2
1 INTRODUCTION
1.1 Air pollution: definition
Air pollution can contain the following substances
[3]:
Gases (e.g. ozone, carbon monoxide,
sulfur oxides, nitrogen oxides)
Organic compounds (e.g. polycyclic
organic hydrocarbons and endotoxins)
Metals (vanadium, nickel and manganese)
PM (particulate matter)
The most health-threatening pollutants are ground-
level ozone and PM [4].
1.2 Particulate matter
Particulate matter can be described as a mixture of
small particles or liquid droplets, consisting of a
number of components like organic chemicals,
acids, metals and soil or dust particles [5].
Depending on their origin, PM can be divided in
primary and secondary particles. The primary
particles originate directly from combustion
processes by several sources like wood combustion,
motorized vehicles and power plants or by
mechanical reduction of coarser materials. The
secondary particles originate from chemical
reactions in the atmosphere [6]. In this article we
use another subdivision, which is based on the
aerodynamic diameter of the particles.
The aerodynamic diameter of a dust particle is the
diameter of a spherical particle that shows the same
behavior in ambient air as the dust particle [7]. PM
is divided in PM10 (≤10µm), PM2.5 (≤2.5µm) and
UFP or ultrafine particulate matter (≤100nm) [8, 9].
The main cause of PM10 are road and agricultural
dust, tire wear, wood combustion and construction
activities [3]. Industrial operations and combustion
are responsible for PM2.5 emission containing e.g.
sulfates, nitrates, carbon, ammonium, hydrogen
ions, lipopolysaccharides (LPS), metals and water.
PM2.5 are originated from oil refineries, the metal
industry, power plants, mobile emission sources,
etc. [3]. Tailpipe emissions from motorized vehicles
are the most important cause of UPF [8-11].
1.3 PM and its health effects
Recently, there has been a growing scientific
interest in the effects of air pollution on human
health [11]. After extensive research, it has become
clear that acute and chronic exposure to PM is
linked to respiratory and cardiovascular health
effects [10, 12]. High ozone concentrations increase
respiratory symptoms in the summer months [13]
and increased wood combustion in winter months
impacts respiratory activity [14]. PM exposure not
only affects respiratory function but it can also
contribute in respiratory and cardiovascular
morbidity and increased total mortality. Chronic
exposure can even be associated with a decreased
life expectancy of 10 years per person [8, 13, 15].
Diesel exhaust particles cause inflammation of the
lungs and thrombosis due to blood platelet
aggregation. Previous studies describe chronic
effects such as an increased risk to develop lung
cancer, COPD, pneumonia, chronic bronchitis and
asthma [16]. Oortgiesen et al. reported a
proinflammatory effect evidenced by the release of
Interleukin 6 (IL6) from bronchial epithelial cells
exposed to PM [17]. IL6 is a cytokine, active in
state of inflammation. IL6 acts also as an anti-
inflammatory cytokine inhibiting the expression of
tumor necrosis factor α (TNFα), which has
proinflammatory functions [18]. In addition, nitric
oxide synthase (NOS) is an enzyme that katalyses
the production of NO which plays an important role
in vasodilatation and bronchodilatation. Three
forms of NOS are investigated in this study.
Neuronal (nNOS/NOS1), inducible (iNOS/NOS2)
and endothelial (eNOS/NOS3). NOS might play a
role in inflammation but the underlying pathways
are still unknown [19, 20]. Thus, inflammation
might play a key role in the negative effects on
human health [10]. Caldéron-Garcidueñas et al.
detected associations between long term PM
exposure and neuroinflammation, blood-brain
barrier (BBB) disruption,cognitive deficits and
brain abnormalities after exposure to air pollution
in children and young adults and in dogs [21]. So
not only peripheral but also in the CNS, cytokine
expression and oxidative stress might occur after
inhalation of ambient PM, ozone and diesel exhaust
[16]. Recently, in a study of suglia et al., an
association was found between PM exposure
cognitive decline in children [22]. . Whereas many
studies focus on systemic inflammation, currently,
researchers focus on the effects of PM inhalation on
the CNS [23]. Circulating cytokines such as TNFα
and IL1β are well known to be able to cross the
BBB and cause neuroinflammation [24].
1.4 Exercise benefits brain function (by
up-regulation of brain-derived
neurotrophic factor (BDNF) levels)
Researchers found that exercise might stimulate
overall human health. Physical activity controls
overweight and combats health conditions and
diseases including high blood pressure,
cardiovascular disease, stroke, metabolic syndrome,
type 2 diabetes, depression, certain types of cancer,
arthritis and falls. In addition, it improves the
mood, boosts energy levels and promotes better
sleep [25]. Previous research revealed that physical
exercise decreases the risk for coronary heart
diseases [26]. The beneficial effects of physical
activity on health go beyond peripheral effects.
Subsequently, recent studies showed beneficial
effects of exercise on neuronal activity, especially
on cognition Exercise was found to improve
learning and memory function in rats [27]. van
3
Praag et al. showed in a study with mice that
voluntary running in a running wheel enhanced
neurogenesis in the dentate gyrus of the
hippocampus, a part that contributes in memory
function [28]. Other studies showed more evidence
for the link between exercise and its benefits in
neuronal function, brain vascularisation, synaptic
plasticity, synaptic transmission and learning [29,
30]. Several studies suggest that exercise stimulates
synaptic plasticity by influencing long-term
potentiation (LTP) and synaptogenesis [28, 31].
Brain vascularisation is the action of creating more
capillaries in the brain [32]. Synaptic plasticity
represents the ability of making connections
between neurons in order to create a better
adaptation power [33, 34]. Chronic stimulation of
neurons results in a long-lasting enhanced signal
transduction between neurons which is called long-
term potentiation (LTP) [35] and synaptogenesis
indicates the making of new neurons [36]. The
main reason for these benefits is found in the
elevated levels of neurotrophic growth factors after
exercise. These factors contribute in the regulation
of survival and differentiation of cells from the
nervous system [37].
Especially BDNF seems to be an important
mediator of the exercise-induced benefits on the
brain. BDNF is a member of the neurotrophin
family. Besides the classical function of regulating
survival (neuroprotection) and differentiation of
neurons [37], it is shown to have an influence on
learning and memory function [38]. It plays a role
in neuronal plasticity and transmission and it has
angiogenic and antidepressant effects [37, 39].
Exercise induces an up-regulation of BDNF in both
animal [28, 40] and human studies [41, 42].
Previous research by Bos et al. revealed up-
regulation of hippocampal BDNF after an exercise
period of 90 minutes [27]. The stimulation of
synaptic plasticity might be induced by the BDNF-
mediated mechanism which involves synaptophysin
(SYP) and synapsin I (SYN1) [31]. SYN1 is
involved in vesicular pool formation and SYP plays
a role in endocytosis [43]. A study of Vaynman et
al. shows the importance of BDNF in this
mechanism by blocking the action of BDNF what
results in no increased levels of SYN1 and SYP
after exercise [43]. Not only BDNF but also
vascular endothelial growth factor (VEGF) and
insulin like growth factor (IGF) might play a role in
the exercise induced brain benefits[39]. IGF-1, a
growth factor primarily produced in the liver, is
important in cell growth and proliferation. It
stimulates neurogenesis in the adult hippocampus
[39]. VEGF, in turn, stimulates angiogenesis. The
more blood vessels, the more nutrients for
neurogenesis [39].
Another purpose for research is to determine the
therapeutic potential of exercise. Again, BDNF
which is linked with improved cognition plays a
key role in this research, [27, 44]. Recent findings
suggest that exercise induced up regulation of
BDNF slows down the process of mental decline
due to aging, accelerates recovery after brain and
spinal cord injury and contributes in preventing or
even cure mental diseases [45]. Research did reveal
that chronic administration of BDNF in humans is
difficult because BDNF might be unable to cross
the blood-brain barrier [46] but this still remains
unclear. A study Boado et al. describes
reformulation of BDNF to a fusion protein as a
solution to cross the BBB [47]. Another study
described the first translocation of BDNF through
the BBB with the use of focused ultrasound and
micro bubbles, a technique to open the BBB [48].
Pending the efficiency, nowadays, a possible way
to intervene in brain recovery, mental disease and
depression could be exercise [45].
The purpose of this study is to investigate the
effects of both exercise and air pollution on the
brain using gene expression analysis. The olfactory
bulbs (OB) are supposed to be the first brain region
to be reached by particulate matter. The OB might
be the most susceptible because of its direct contact
with the outside. In this tissue we are searching for
effects of PM exposure. Hippocampus and
prefrontal cortex are selected for analysis because
they are critical regions for cognition. Because the
analysis of brain tissue still remains an invasive
technique, we also have taken blood samples as an
alternative analysis. The purpose is to translate
hippocampal inflammatory and neurothrophic
effects to the blood.
2 EXPERIMENTAL SET UP
2.1 Rat experiment (Exercise and UFP)
In the first experiment, 24, male albino rats, coming
from Charles River (Germany), weighing 175-200g
were housed socially in a 12-h light-dark room for
6 days before starting treadmill habituation. The
rats had ad libitum access to food and water. All
experiments were run with the approval of the
Ethics Committee on Animal Experiments of the
Faculty of Medicine and Pharmacy of the Vrije
Universiteit Brussel.
4
All animals had a habituation period on a motor
driven horizontally treadmill, one week before the
experiment. During this week, the speed and
duration of exercise were increased until the
animals reached the level of 75 min of 20 m/min.
Next, the animals were divided into groups of 6
animals (Ambient air-Rest = Control group,
Ambient air-Exercise = group B, UFP – Rest =
group C, UFP-Exercise = group D). Subsequently,
the rats ran or rested for 90 minutes in either
ambient air or UFP polluted air. At last, the rats
were euthanized 24h after the exposure. Three
Brain regions (OB, prefrontal cortex and
hippocampus) were collected and put on
RNAlater® (Life Technologies, Ghent, Belgium) to
stabilize the RNA, at 4°C overnight. Next,
RNAlater® was removed and the samples were
stored at -80°C. Also, blood samples were taken
24h after the exposure and kept on RNAlater® at -
80°C. In order to understand which of the examined
genes are up or down regulated in test and/or
control groups, the gene expression changes in rat
brain tissue were previously studied for a limited
set of genes [27]. The goal is to search for
expression changes in a larger set of genes in brain
and blood samples of the rats.
The administration of UFP, as seen in Fout!
Verwijzingsbron niet gevonden., was given by the
mini Combustion Aerosol Standard (miniCAST)
model 6203-A (Jing Ltd, Zollikofen, Switzerland).
The machine expels 3L/min particles soot. The
reader is referred to Bos et al., 2012 for more
details on study design [27].
2.2 Mouse experiment
(Craeybeckxtunnel)
In the second experiment, 20 male, 6 week-old
C57BL6 mice were put into a busy highway tunnel
in Antwerp (Craeybeckxtunnel, Figure ). The
animals had ad libitum access to food and water.
During the experiment, one group of five mice was
put into a polyethylene cage without filtercap (Test
group) and a second group of five mice in a cage
with filtercap (Control group A). Control group B
was placed outside the tunnel in a nearby building
and the last control group was placed in an animal
house in Leuven. Control group A was included to
discriminate between effects of PM exposure and
effects of stress due to noise inside the tunnel
environment.
Figure 2: Craeybeckxtunnel Antwerp [57]
Air quality was measured inside the tunnel with a
low volume filter sampler (Partisol, Thermo). The
filters (type Tissue Quartz) were analyzed in the lab
for elemental carbon (EC) and organic carbon (OC)
by Thermo Optical Transmission (TOT, Sunset)
and the NIOSH protocol (Peterson & Richards,
2002). A Scanning Mobility Particle Sizer (TSI
SMPS, Model 3936) measured total particle
number and UFP size. A handheld CPC (P-Trak,
TSI, 20nm – 1000nm) was used to measure
concentrations inside and outside the cages in the
tunnel. After the exposure, the lungs of the animals
were removed to investigate characteristics of
inflammation [49]. Next, the hippocampus and the
bulbus olfactorius were removed for further gene
expression analysis. The tissue was kept overnight
at 4°C on RNAlater®. Then, the RNAlater® was
removed by decantation and the samples were
stored at -80°C. Different brain tissues of these
animals were already examined for a small set of
genes [49]. The purpose of this study is to examine
a larger set of genes in samples from the mouse
brain. A small amount cDNA is already available
for qPCR analysis. The reader is referred to Bos et
al., 2012 for more details on study design [27]. The
effect of a short PM exposure on the mouse brain
will be investigated at the gene expression level
looking at changes in the expression of
inflammatory genes (NOS1), and neurotrophic
factors (BDNF, VEGF and IGF1). Table 1 gives an
overview of the selected genes.
2.3 RNA extraction from blood
RNA extractions from whole rat blood were
performed using the Mouse RiboPure™ - Blood
RNA Isolation Kit (Ambion) according to the
manufacturer’s instructions. Blood samples were
thawed and centrifuged before the extraction
procedure was started. The RNA isolation is based
Figure 1: Experimental installation with
Minicast, UFP monitor and CO2-monitor [49]
5
on cell lyses followed by a phenol-chloroform
extraction and a purification step using the glass
fiber filter technology. The concentrations of the
RNA samples were measured using the Nanodrop®
Spectrophotometer (Isogen, Life Science, De
Meern, the Netherlands). The samples were stored
at -80°C.
2.4 cDNA synthesis
The cDNA synthesis out of total RNA was
performed using the Transcriptor First Strand
cDNA Synthesis kit of Roche (Vilvoorde, Begium).
The concentrations of the RNA samples, extracted
from rat blood and hippocampus were measured in
order to calculate the exact volume per sample to
become a sample of 1 µg/20µl. The calculations are
shown in Appendix 1 and Appendix 2. The cDNA
synthesis reactions were executed with both
hexamer primers and anchored oligo dT primers.
The reactions were performed in duplicate using
Verity 96-well Thermal Cycler of Applied
Biosystems (Life Technologies). One µg RNA was
estimated to yield 1 µg cDNA, becoming a cDNA
concentration of 1 µg/20µl.
2.5 QPCR
The qPCR reactions were run in triplicate using a
LightCycler 480 Probes Mater kit (Roche) and
PrimeTime Mini qPCR Assay (Integrated DNA
Technologies). The samples were diluted with
nuclease free water to become a concentration of 4
ng/µl. Aliquots of 20 µl were obtained in 96-well
plates for all qPCR amplification reactions in the
Roche LightCycler 480. Each reaction mixture
contains 5 µl of DNA sample and 15 µl master mix.
This mix consists of 4 µl nuclease free water, 1 µl
20X PrimeTime assay and 10 µl 2X concentrated
Master Mix. qPCR program consists of a
preincubation cycle (10 min at 95°C), 45
amplification cycles (10 sec at 95°C, 30 sec at 62°C
and 1 sec at 72°C) and a last cooling cycle (10 sec
at 40 °C). The analysis output calculates sample
specific crossing points using the “Second
Derivative Maximum Method”.
2.6 Statistical analysis
Statistical analysis was performed using the
Relative Expression Software Tool (REST© 2009
V.2.0.13; [50]). The software compares 2 groups,
one control group and one experimental group. All
qPCR reactions were performed in triplicate with a
variance maximum between technical replicates of
0.8. Expression stability of putative housekeeping
genes was verified using geNorm [51]. All qPCR
reactions were considered 100% efficient. The
program calculates the normalized relative gene
expression ratio (R) using the formulae shown in
appendix 3. Subsequently, the statistical
significance is calculated using the Pair Wise Fixed
Reallocation Randomization Test© with 10,000
permutations. The Pair Wise Fixed Reallocation
Randomization Test© is a valid statistical test for
gene expression analysis which is based on
permutation analysis [50]. The test does not make
assumptions about the distribution of data while it
remains as powerful as parametric tests [49]. For
more detailed information, the reader is referred to
a paper of Pfaffl et al [50].
6
Table 1: Overview of the genes selected for analysis
Full name (gene/protein)
Abbreviation
Rat
blood
Rat brain (H, PFC, OB)
Mice brain(H + OB)
Reference genes
β-2 Microglobulin
B2M
x
X (Only hippocampus extra)
Glyceraldehyde-3-phosphate dehydrogenase
GAPDH
x
X (Only hippocampus extra)
Hypoxanthine phosphoribosyltransferase 1
HPRT1
x
X (Only hippocampus extra)
Tyrosine 3-monooxygenase/tryptophan 5-
monooxygenase activation protein, zeta polypeptide
YWHAZ
x
X (Only hippocampus extra)
Target genes (Bos et
al. [27, 49])
Brain-Derived Neurotrophic Factor
BDNF
x
X (Only hippocampus extra)
Nuclear factor, erythroid-derived 2, like 2
NFE2L2
x
Interleukin 1α
IL1α
x
Interleukin 1β
IL1β
x
Interleukin 6
IL6
x
Tumor Necrosis Factor
TNF
x
Inducible NO-synthase
NOS2 (iNOS)
x
Endothelial NO-synthase
NOS3 (eNOS)
x
Target genes
Neuronal NO-synthase
NOS1 (nNOS)
x
x
x
Insuline-like Growth factor 1
IGF1
x
x
x
Vascular Endothelial Growth Factor
VEGF
x
x
x
Synapsin 1
Syn 1
x
x
Synaptophysin
Syp
x
x
Prostaglandin-endoperoxide synthase 2 (also: Cyclo-
oxygenase 2, COX-2)
COX2 (rat)
x
x
Target genes (Extra)
Tyrosine kinase B receptor
TrkB r
x (Only Hippocampus)
calcium/calmodulin dependant protein kinase 2a
CAMK-II
x (Only Hippocampus)
cAMP response-element-binding
CREB
x (Only Hippocampus)
Mitogen-activated protein kinase
MAP-K
x (Only Hippocampus)
Uncoupling protein 2
UCP2
x (Only Hippocampus)
Growth Associated protein 43
GAP43
x (Only Hippocampus)
Legend: Genes indicated with the X are tested on the selected samples.
7
0
0,5
1
1,5
2
2,5
3
IGF1
NOS1
VEGFa
Expression ratio
Mice Hippocampus
Hippocampus: Test
group vs control B
Hippocampus: Control A
vs control B
Hippocampus: Test
group vs control A
**
##
Figure 3: Hippocampal gene expression of selected genes. Data indicate the expression in the test group
in the tunnel relative to control B in the building (black bars), the expression in control A in the tunnel
with filter cap relative to control B (white bars) and the expression in the test group relative to control
A (gray bars). Error bars indicate standard errors, n = 5 for each treatment. Significance is indicated
for Test group versus control B (*p<0.05; **P<0.01), Test group versus Control A (#p<0.05; ##p<0.01).
IGF1, interleukin like growth factor 1; NOS1 nitric oxide synthase 1; VEGFa, vascular endothelial
growth factor a.
0
2
4
6
Il1b
NOS2
NFE2L2
Tnf
Il6
NOS3
NOS3_2
Ptgs2
VEGF
IGF1
SYN1
Expression ratio
Rat Blood
UFP/Rest
Ambient
air/Exercise
UFP/Exercise
*
*
*
$
#
#
Figure 4: Blood gene expression of selected genes. Data are expressed relative to the control – Group
(Ambient air/Rest). Error bars indicate standard errors, n = 6 for each treatment. Significance is
indicated for UFP/Rest (*p<0.05; **P<0.01), for Ambient air/ Exercise ($p<0.05; $$p<0.01) and for
UFP/Exercise (#p<0.05; ##p<0.01). IL1b, interleukine 1 b; NOS2, nitric oxide synthase 2; NFE2L2,
Nuclear factor, erythroid-derived 2, like 2; Tnf, tumor necrosis factor; IL6, interleukin 6; NOS3, nitric
oxide 3; NOS3_2, nitric oxide synthase 3 (2nd test); Ptgs2, Prostaglandin-endoperoxide synthase 2 (also:
COX-2, Cyclo-oxygenase 2); VEGF, vascular endothelial growth factor; IGF1, interleukin like growth
factor 1; SYN1, synapsin 1.
8
3 RESULTS
3.1 Gene expression analysis
3.1.1 Mice hippocampus
The qPCR analysis was reliable with technical
replicates varying less than 0.8 Cp units. The
detection limit was set on a Cp value up to 40
cycles. The maximum detected Cp value in the
hippocampus group was 30.37 cycles which was
below maximum detection limit. Significant
changes in the hippocampus were visible in 1 out of
3 assays. Nitric oxide synthase 1 shows a
significant up regulation in the hippocampus in the
test group (expression ratio (R) = 1.38; p < 0.01)
compared to control A and in the test group (R =
1.75; p < 0.01) compared to control B1. Results are
shown in figure 3.
3.1.2 Rat blood
In the rat blood, five out of sixteen assays were
close to the detection limit 40. Therefore a Cp value
of 38 was set as threshold value. All Cp values
higher than 38 were excluded for statistical
analysis. When two or three out of three technical
replicates were excluded, the sample was discarded.
1 Gene expression analysis for the mice olfactory bulb did not
show decisive results and are not described in the results. Graphs
can be found in appendix 4
The minimum number of samples per group was set
to four, whereby treatment groups with less than
four samples were not analyzed by REST. All
treatment groups were compared to the control
group (rest/ambient air) in statistical analysis.
Further, three out of eleven assays showed a
significant up regulation in rat blood and one
showed down-regulation. Nuclear factor (erythroid-
derived 2)-like 2 (NFE2L2) was up regulated (R =
1.87; R < 0.05) in the UFP/Exercise group. In the
same group a down regulation (R = 0.18; R < 0.01)
was observed for NOS3. The UFP/REST group
showed a significant increase in expression of
TNFα (R = 1.50; p < 0.05) and SYN1 (R = 2.25; p
< 0.05) and a down regulation of NOS3 (R = 0.086;
p < 0.05). IL1β was down regulated (R = 0.73; p <
0.05) in the control exercise group (figure 4). A
trend towards up regulation was found for TNFα
(R= 0.072; p < 0.1) in the UFP/exercise group.
3.1.3 Rat hippocampus
All treatment groups were compared to the control
group (ambient air /rest). Previous research by Bos
et al. showed a significant up-regulation of BDNF
(R = 1.543; R < 0.05) in the Ambient air/exercise
group and a trend to down-regulation (R = 0.657; p
= 0.059) in the UFP/Rest group. Further analyses of
the selected genes showed significant down-
regulation of 3 assays out of 4 and no significant up
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
2
BDNF
SYN1
VEGFa
Ptgs2
IGF1
Expression ratio
Rat Hippocampus
UFP/Rest
Ambient air/Exercise
UFP/Exercise
##
*
$
**
#
**
$$
#
Figure 5: Hippocampal gene expression of selected genes. Data are expressed relative to the control-
group (ambient air/rest). Error bars indicate standard errors, n = 6 for each treatment. Significance is
indicated for UFP/Rest (*p<0.05; **P<0.01), for Ambient air/ Exercise ($p<0.05; $$p<0.01) and for
UFP/Exercise (#p<0.05; ##p<0.01). BDNF, brain-derived neurotrophic factor; SYN1, synapsin 1; VEGFa,
vascular endothelial growth factor a; Ptgs2; Prostaglandin-endoperoxide synthase 2 (also: COX-2,
Cyclo-oxygenase 2); IGF1, interleukin like growth factor 1.
9
regulations. In the UFP/Rest group down-regulation
of SYN1 (R = 0.672; p < 0.05), VEGFa (R = 0.408;
p < 0.01) and prostaglandin-endoperoxide synthase
2 also known as cyclooxygenase 2 (COX2) (R =
0.529; p < 0.01) was found. A down regulation (R =
0.079; R < 0.01) of Ptgs2 was found in the exercise
group in ambient air. SYN1 (R = 0.668; p < 0.01)
showed a decreased expression in the UFP exercise
group. Similarly, the expression of VEGFa (R =
0.684; p < 0.05) and Ptgs2 (R = 0.649; p < 0.05)
was down regulated in the UFP/Exercise group2
(Figure 5).
4 DISCUSSION
Two animal studies were set up to investigate the
effects of both air pollution and physical activity on
the gene expression of inflammatory markers,
neurotrophic growth factors, proteins important in
neurotransmission & synaptic plasticity in the
brain. In a mice experiment the mice were put into
a busy highway tunnel in Antwerp (Craeybeckx
tunnel). We selected the OB to investigate because
this might be the first brain region to be reached by
PM [52, 53]. Hippocampal tissue was also analyzed
in both studies because of its importance in learning
and memory [54]. The samples of the OB did not
show consistent results, we, therefore, mainly
focused on hippocampal results. In the second
experimental set up, rats were 90 min exposed to
UFP or ambient air while running or resting on a
treadmill. Twenty-four hours after exposure, rats
were sacrificed in order to examine gene expression
changes in blood and brain tissue including
hippocampus, OB and prefrontal cortex. The
prefrontal cortex was chosen for its contribution in
goal-direction, complex human behavior and
working memory [55]. OB and prefrontal cortex
analysis did not show consistent results. The focus
in this research was laid on hippocampal tissue.
In the mice experiment, previous research of Bos et
al. did not show pulmonary or systemic
inflammation but gene expression changes were
detected [49]. Previous research of Bos et al.
already revealed air pollution induced up regulation
of COX2, NOS2 and NOS3 in the hippocampus
[49]. In addition, we found significant changes in
the expression of NOS1, which is an important
inflammatory response gene [54], after an exposure
of 5 days to high levels of air pollution. In both test
group versus control A and test group versus
control B, NOS1 was significantly up-regulated.
The comparison of both control groups did not
show a significant effect. This combination was
2 Gene expression analysis for the rat olfactory bulb and
prefrontal cortex did not show decisive results and are not
described in the results. Graphs can be found in appendix 5 and
6
taken into account to exclude stress factors like
noise inside the tunnel. These results correspond to
observations of Jang et al. in an ozone exposure
study in mice who also found an increased
expression of NOS1 after exposure to ozone
polluted air [54]. The previous study by Bos et al.
also showed increased expression of NOS2 and
NOS3, as a result of air pollution [49]. In a cohort
study of Caldéron-Garcidueñas et al. an increased
mRNA expression of COX2 was found, due to air
pollution in dogs [56]. Another article refers to
increased expression of NOS2 after chronic
exposure to air pollution in dogs [57]. Chronic
inflammation plays a key role in neuronal death,
associated with neurodegenerative diseases like
Alzheimer’s disease (AD) and Parkinson [58-61].
Since it is known that not only BDNF plays a role
in brain plasticity, but also VEGFa and IGF1 [39],
we chose to investigate these neurotrophic growth
factors in addition. The expression levels of both
growth factors IGF1 and VEGFa did not change
significantly in contrast to the BDNF gene
expression which was decreased in the OB, shown
previously in Bos et al. [49]. We were the first to
investigate the effects of air pollution on
hippocampal mRNA levels of VEGFa and IGF1.
VEGFa contributes in angiogenesis, neurogenesis,
neuroprotection and astroglial proliferation as
shown in figure 6. Brain vascularisation increases
due to exercise as a result of increased mRNA
resulting in increased VEGFa protein levels (Figure
6) [20]. IGF-1, a growth factor primarily produced
in the liver, is important in cell growth and
Figure 6: VEGFa functions in a schematic
overview [1]
10
proliferation. It stimulates neurogenesis in the adult
hippocampus [39].
In the rat study, we examined not only the effects of
polluted air but also the effects of physical activity.
Bos et al. was the first to investigate the effects of
physical activity in combination with air pollution
on the brain. In agreement with previous studies
[REF], Bos et al. showed a significant up-regulation
of BDNF in hippocampal tissue as a result of
exercise in ambient air. The effect was not present
in the exercise group in combination with UFP
exposure [27]. Not only BDNF but also several
inflammatory genes were affected by exercise and
UFP exposure in different brain regions [27]. As a
sequel to this study, we investigated a wider range
of inflammatory, neurotrophic growth factor and
synaptic plasticity related genes. Synapsin 1, a
protein regulating vesicular release at the synapse
which is important for neurotransmission and
synaptic plasticity, showed the same pattern as
BDNF but was not significantly increased due to
exercise in ambient air.
Since exercise increases BDNF levels, the positive
effects of physical activity might be attributed to
the BDNF mediated mechanism [2]. Exercise is
shown to have an influence on learning and
memory function [38]. Recent findings suggest that
exercise slows down the process of mental decline
due to aging, accelerates recovery after brain and
spinal cord injury and contributes in preventing and
slowing down the progress of neurodegenerative
diseases like Alzheimer [62, 63] and Parkinson [35,
62]. Also stress levels can be reduced due to
exercise resulting in an anti-depressant effect [45].
In contrast, SYN1 was down regulated in both
UFP/rest and UFP/exercise groups which might
suggest a decreased synaptic plasticity and
neurotransmission after exposure to UFP. Yet, little
research about SYN1 is done but Baldelli et al.
suggested that SYN1 is responsible for the
translocation of neurotransmitter vesicles from the
reserve pool towards the ready release pool. A lack
of SYN1 can result in a shortage of
neurotransmitter release and, thus a decreased
signal transmission [64].
Not only SYN1 but also VEGFa was down
regulated after UFP exposure regardless of whether
physical activity was performed or not. As
described in the previous paragraph, the UFP
induced down-regulation was not visible in the
mice experiment but is visible in the rat experiment.
SYN1 showed mainly the same pattern as BDNF
which is in support with previous studies
Figure 7: Potential BDNF mediated mechanism. BDNF is responsible for the energy independent
mechanism which influences learning and memory by neuro - and synaptogenesis. It influences important
pre-synaptic and post-synaptic cascade reactions [2].
11
demonstrating a key role of BDNF in the exercise-
induced enhancement of brain plasticity & function.
This BNDF-mediated mechanism is thought to
follow the pathway as shown in figure 7. Although
it is known that gene expression is a very dynamic
process, yet, in this research we investigated the
expression of other genes, suggested to be involved
in this pathway, such as the Tyrosine kinase B
receptor, cAMP response-element-binding (CREB)
protein, calcium/calmodulin dependant protein
kinase 2a (CAMKIIa), nitric oxide synthase 1
(NOS1), Syntaxin 11 (SYT11), Growth Associated
protein 4 (GAP4) and uncoupling protein 2 (Ucp2)
whose expression may also be affected. CREB is a
transcriptional regulator [43] and UCP2 is
hypothesized to be an upstream mediator of
exercise induced effect on BDNF. Blocking of the
UCP2 expression results in no exercise induced up-
regulation of BDNF, SYN1, SYP, etc. [2, 65].
The increased BDNF levels in response to exercise
also stimulate Gap4 which is present in growing
axon terminals. It plays a role in synaptic plasticity
and is implicated in learning and memory function
[66]. Exercise also increases levels of CAMKII,
which is a protein of a signal transduction pathway
that is activated in response to exercise [67]. Also
the BDNF receptor TrkB is involved in the
mechanism. Up-regulation of BDNF might
stimulate a positive feedback loop increasing its
own levels as well as those of TrkB [2]. Results are
shown in Appendix 7.
Furthermore we observed a significant decrease of
COX2 gene expression in the UFP/REST,
AmbientAir/Exercise and UFP/Exercise groups.
Cyclo oxygenase 2 is an important inflammatory
marker [68]. Chronic physical activity shows a
clear anti-neuroinflammatory effect in the brain
[27] but acute bouts of exercise induce a short pro-
inflammatory response. Twenty-four hours after the
activity this response is already gone. An acute
physical action can be seen as a stressful
stimulation on what the body reacts with a pro-
inflammatory response [69]. Still there is little
research concerning the effects of exercise on
neuroinflammation. Literature still remains unclear
about possible effects. In this study we found a
down regulation in both cases which was partially
in accordance with our hypothesis. In our study, the
acute bout of exercise induced down regulation of
inflammatory markers. In this study, not only acute
physical activity but also exposure to UFP could
evoke down regulation of COX2 expression.
However the effect of UFP/Exercise was thought to
be bigger. A study of Caldéron-Garcidueñas et al.
suggests that the pathology of AD is characterized
by brain inflammation. A pollution mediated up
regulation of COX2 and IL1β might contribute in
the development of AD and other
neurodegenerative diseases [68].
Investigation of brain tissue still remains an
invasive technique. Therefore the use of blood
samples might be an important solution to avoid the
sacrifice of laboratory animals. For this reason we
investigated inflammatory genes as well as genes
with a known function in neural plasticity in blood
samples, taken 24 hours after the rat experiment.
Sixteen genes were analyzed of which 11 are shown
in Figure 4. Detection limit for Cp values is 40 but
was set on 38 to create a comfortable zone between
undetectable and detectable values. 4 out of 16
genes were excluded because of unreliability and
values higher than the detection limit. At first, an
exercise-induced down regulation of inflammatory
marker IL1β and an UFP/exercise induced up-
regulation of NFE2L2 was observed. Also a trend
towards up regulation of TNFα was observed in the
UFP/exercise group. It is clear that the expression
of TNFα and NFE2L2 increases with UFP exposure
regardless their physical activity. This is consistent
with previous findings. An important oxidative
stress related transcription factor is NFE2L2, which
increases the expression of several antioxidant
enzymes in response to oxidative stress [70-72].
Acute exercise is an acute stressor which might
elevate mRNA levels of inflammatory markers [73-
75]. Chronic exercise, in turn, might result in a
down regulation of inflammatory genes [74, 76].
Overall chronic exercise has beneficial effects on
inflammatory and oxidative stress in the brain [69,
77] and blood [78]. In addition, a decreased
expression of NOS3 is observed in both UFP
groups, regardless their activity. These findings do
not follow the hypothesis of increased
inflammatory response after exposure to air
pollution. Nitric oxide synthase 3 is involved in
regulation of vasoconstriction. It generates nitric
oxide in blood vessels which stimulates
vasoconstriction [20] as shown in figure 8. At last a
significant increase of SYN1 was found in the
UFP/Rest group. These finding do not stroke with
our hypothesis. In the rat hippocampus an exercise
induced trend towards up regulation SYN1 was
seen.
12
A possible explanation for the lack of inflammatory
effects, excluding TNFα, in this study might be the
relative long period between exposure and sacrifice.
We have chosen to analyze tissue and blood 24
hours after the exposure according to the research
of Gerlofs-Nijland et al. [79]. In their study the
most important health effects were observed 24
hours after the exposure [79]. BDNF might also be
affected in an earlier stage after the exposure [80].
Gene expression is a dynamic process that may be
underlying the differences in expression between
several related genes. Therefore, in the future, it
might be better to implement an examination of the
effects at different time intervals in order to
uncover the dynamic character of gene expression.
Another problem was the low expression level of
several genes in the blood. Brain related genes, in
particular, showed low gene expression levels in
blood. Moreover, we could not detect expression of
BDNF in rat blood. A study of Hashimoto et al.
shows detectable mRNA levels of BDNF in the
human bloodstream [81]. Another study showed
detectable gene expression of BDNF in mice blood
[82]. These findings might entail detectable levels
of BDNF gene expression in blood. In our study we
expected to observe air pollution mediated up
regulation of inflammatory genes in both
hippocampus and bloodstream. Our findings do not
stroke with this hypothesis.
BDNF was tested in the previous study of bos et al.
[27] where an exercise induced up regulation was
found. In order to test more genes, involved in the
BDNF-mediated mechanism, a new batch of cDNA
was made. In effort to make both batches
comparable, BDNF was tested on batch two. In this
case, BDNF was no longer up regulated and the
other implicated genes did not show decisive
results. The results are shown in Appendix 7. No
real failures in technical replicates of both lab
technicians were found which excludes pipetting
errors. The use of different interpolate calibrators
(same gene combined with the same sample on
each plate) excludes deviations between different
plates. The most obvious reason might be the easy
degradation of RNA after being thawed and
refrozen for several times.
5 CONCLUSIONS
Previous results of this study did already reveal
negative inflammatory effects of air pollution in the
hippocampus of mice and a decreased neurotrophic
support in the OB of the mice. Our findings
correspond to previous results of Bos et al.. Despite
that more evidence has been provided for the
hypothesis of brain inflammation, evoked by
polluted air, no other negative effects on synaptic
plasticity related genes could be observed. However
we also found in the mice experiment inflammatory
effects of air pollution on hippocampus, these
findings are not translated to effects in the rat
hippocampus. Nevertheless this study does reveal
decreased expression of plasticity related genes due
to UFP in the rat hippocampus. Despite these
findings in the hippocampus, none of the effects
could be observed in blood samples.
Figure 8: eNOS generates NO in endothelial cell.
Acetylcholine stimulates Ca2+ influx in the cell
which contributes in the NO production.
Increased NO results in vasoconstriction [20].
13
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16
7 APPENDICES
Appendix 1: cDNA synthesis Rat blood
cDNA synthesis: Rat Blood
Nr.
Sample
Conc.
ng/ul
Sample
1µg ( µL)
Anchored dT
Primers (µL)
Hexamer
primers (µL)
H2O until
10 µl
Incubat
ion:10
min
65°C
RT mix
(µL)
1
E1
544,62
1,8
1
2
8,2
7
2
E2
203,75
4,9
1
2
5,1
7
3
E3
327,69
3,1
1
2
6,9
7
4
E4
272,88
3,7
1
2
6,3
7
5
E5
283,07
3,5
1
2
6,5
7
6
E6
284,05
3,5
1
2
6,5
7
7
E10
227,62
4,4
1
2
5,6
7
8
E11
177,76
5,6
1
2
4,4
7
9
E12
192,62
5,2
1
2
4,8
7
10
E7
409,36
2,4
1
2
7,6
7
11
E8
320,16
3,1
1
2
6,9
7
12
E9
282,53
3,5
1
2
6,5
7
13
CE24h1
384,31
2,6
1
2
7,4
7
14
CE24h2
297,89
3,4
1
2
6,6
7
15
CE24h3
516,43
1,9
1
2
8,1
7
16
CE24h5
332,82
3,0
1
2
7,0
7
17
CR24h3
604,94
1,7
1
2
8,3
7
18
CR24h4
403,17
2,5
1
2
7,5
7
19
CE24h6
253,85
3,9
1
2
6,1
7
20
CR24h5
259,13
3,9
1
2
6,1
7
21
CR24h6
178,16
5,6
1
2
4,4
7
Each samples was performed in
double
Mix (48
reactions
)
RT
buffer
(µL)
Rnase inh
(µL)
Deoxynucleotide
mix (µL)
RT (µL)
192
24
96
24
17
Appendix 2: cDNA synthesis Rat hippocampus
cDNA synthesis: Rat hippocampus
Nr
Staal
Conc
ng/ul
Staal 1
µg ( µL)
Anchored dT
Primers (µL)
Hexamer
primers (µL)
H2O
until 10
µl
10min
65°C
RT
mix
(µL)
1
E1H
272,32
3,7
1
2
6,3
7
2
E2H
291,37
3,4
1
2
6,6
7
3
E3H
251,25
4,0
1
2
6,0
7
4
E4H
284,77
3,5
1
2
6,5
7
5
E5H
241,53
4,1
1
2
5,9
7
6
E6H
204,77
4,9
1
2
5,1
7
7
E7H
236,95
4,2
1
2
5,8
7
8
E8H
241,14
4,1
1
2
5,9
7
9
E9H
99,42
10,1
1
2
-0,1
7
10
E10H
147,4
6,8
1
2
3,2
7
11
E11H
123,84
8,1
1
2
1,9
7
12
E12H
229,77
4,4
1
2
5,6
7
13
CE24h1
229,14
4,4
1
2
5,6
7
14
CE24h2
327,9
3,0
1
2
7,0
7
15
CE24h3
203,71
4,9
1
2
5,1
7
16
CE24h4
160,46
6,2
1
2
3,8
7
17
CE24h5
187,93
5,3
1
2
4,7
7
18
CE24h6
231,89
4,3
1
2
5,7
7
19
CR24h1
315,09
3,2
1
2
6,8
7
20
CR24h2
314,27
3,2
1
2
6,8
7
21
CE24h3
185,65
5,4
1
2
4,6
7
22
CR24h4
236,94
4,2
1
2
5,8
7
23
CR24h5
240,2
4,2
1
2
5,8
7
24
CR24h6
222,27
4,5
1
2
5,5
7
Each samples was performed in
double
RT
buffer
(µL)
Rnase
inh (µL)
Deoxynucleotidemix
(µL)
RT (µL)
Mix (48
reactions)
216
27
108
27
Appendix 1 and 2: After the RNA extraction, concentrations of RNA samples were measured in order to define
the right concentration for the cDNA synthesis. For example: The first concentration was 544.62 ng/ul. 1 µg is
needed in this reaction to have a final concentration of 1µg/20µL cDNA. Therefore 1,8 µl is needed. This 1.8 µl
needs to be diluted to 10µl. The protocol prescribes for each aliquot 1µl Anchored oligo dT primers and 2 µl
hexamer primers. Afterwards, an incubation period of 10min. at 60°C is needed hybridize the primers. At last
7µl RT-mix is added to become a total volume of 20µl for the PCR reaction.
18
Appendix 3: Equation for the calculation of the relative expression ratio
With n the number of housekeeping genes
Appendix3: The calculation of the relative expression ratio is based on a normalization of the Cp values with the
values of the housekeeping genes. First al housekeeping genes are recalculated to a geometric mean. Than the
target genes are compared to this geometric mean in order to be expressed as a relative expression ratio
Appendix 4: Mice OB results
Appendix 4: OB gene expression of selected genes. Data indicate the expression in the test group in the
tunnel relative to control B in the building (black bars), the expression in control A in the tunnel with
filter cap relative to control B (white bars) and the expression in the test group relative to control A (gray
bars). Error bars indicate standard errors, n = 5 for each treatment. Significance is indicated for control
A versus control B ($p<0.05). IGF1, interleukin like growth factor 1; NOS1 nitric oxide synthase 1;
VEGFa, vascular endothelial growth factor a.
Appendix 4: In the mice OB, VEGFa is significantly down regulated (R= 0.649; P=0.018) in the control A
group compared with control B.
0,000
0,500
1,000
1,500
2,000
2,500
3,000
3,500
4,000
4,500
5,000
IGF1
NOS1
VEGFa
Expression ratio
Mice Olfactory Bulb
OB: Test group vs
control B
OB: Control A vs
control B
OB: Test group vs
control A
$
19
Appendix 5: Rat OB results
Appendix 5: OB gene expression of selected genes. Data are expressed relative to the control-group
(ambient air/rest). Error bars indicate standard errors, n = 6 for each treatment. Significance is indicated
for UFP/Rest (*p<0.05; **P<0.01). BDNF, brain-derived neurotrophic factor; SYN1, synapsin 1; VEGFa,
vascular endothelial growth factor a; Ptgs2; Prostaglandin-endoperoxide synthase 2 (also: COX-2, Cyclo-
oxygenase 2); IGF1, interleukin like growth factor 1; NOS1, nitric oxide synthase 1; SYP, synaptophysin.
Appendix 5: In the OB of the rats a significant down regulation was seen of VEGFa (R= 0.689; P= 0.006) due
UFP by animals in rest compared with the Ambient air/Rest group. This result strokes with the hypothesis of
decreased neurotrophic support due to UFP but there are no other interesting evidences that confirm the
hypothesis.
0
0,5
1
1,5
2
2,5
BDNF
SYN1
VEGFa
Ptgs2
IGF1
NOS1
SYP
Expression ratio
Rat OB
UFP/Rest
Ambient air/Exercise
UFP/Exercise
**
20
Appendix 6: Rat prefrontal cortex results
Appendix 6: PFC gene expression of selected genes. Data are expressed relative to the control-group
(ambient air/rest). Error bars indicate standard errors, n = 6 for each treatment. Significance is indicated
for UFP/Rest (*p<0.05; **P<0.01), for Ambient air/ Exercise ($p<0.05; $$p<0.01) and for UFP/Exercise
(#p<0.05; ##p<0.01). BDNF, brain-derived neurotrophic factor; IL1a, interleukin 1 a; IL1b, interleukine 1
b; NFE2L2, Nuclear factor, erythroid-derived 2, like 2; NOS2, nitric oxide synthase 2; NOS3, nitric oxide
3; Tnf, tumor necrosis factor; SYN1, synapsin 1; VEGF, vascular endothelial growth factor ;Ptgs2,
Prostaglandin-endoperoxide synthase 2 (also: COX-2, Cyclo-oxygenase 2); IGF1, interleukin like growth
factor 1.
Appendix 6: In the prefrontal cortex of the rats, a down regulation was observed of IL1α (R= 0.774; P=
0.046), NOS3 (R= 0.683; P= 0.047), SYN1 (R= 0.625; P= 0.001) and VEGFa (R= 0.758; P= 0.027) due to
exercise in ambient air. IL1α and NOS3 were examined in previous research done by Bos et al. [27] and are
inflammatory genes. SYN1 and VEGFa are brain plasticity related genes. These results do not show decisive
effects.
0
0,5
1
1,5
2
2,5
BDNF
IL1α
IL1β
NFE2L2
NOS2
NOS3
TNF
SYN1
VEGFa
Ptgs2
IGF1
Expression ratio
Rat PFC
UFP/Rest
Ambient air/Exercise
UFP/Exercise
$
$
##
##
$
$$
21
Appendix 7: Rat Hippocampus results
Appendix 7: Hippocampal gene expression of selected genes. Data are expressed relative to the control-group (ambient air/rest). Error bars indicate standard
errors, n = 6 for each treatment. Significance is indicated for UFP/Rest (*p<0.05; **P<0.01), for Ambient air/ Exercise ($p<0.05; $$p<0.01) and for UFP/Exercise
(#p<0.05; ##p<0.01). BDNF, brain-derived neurotrophic factor; ; IL1a, interleukin 1 a; IL1b, interleukine 1 b; NFE2L2, Nuclear factor, erythroid-derived 2, like 2;
NOS2, nitric oxide synthase 2; NOS3, nitric oxide 3; Tnf, tumor necrosis factor; SYN1, synapsin 1; VEGFa, vascular endothelial growth factor a; Ptgs2;
Prostaglandin-endoperoxide synthase 2 (also: COX-2, Cyclo-oxygenase 2); IGF1, insulin like growth factor 1; NOS1, nitric oxide synthase 1; SYP, synaptophysin;
CREB, cAMP response-element-binding protein; TRK2, Tyrosine kinase B receptor; BDNF, brain-derived neurotrophic factor; SYT11, syntaxin11; UCP2,
uncoupling protein 2; CAMK2a, calcium/calmodulin dependant protein kinase 2a; GAP43, Growth Associated protein 43.
The ultimate goal of the study
was to try to investigate the
BDNF-mediated mechanism.
Therefore we needed more
cDNA. Because the previous
batch of cDNA wasn’t
enough, we were forced to
make a new batch. These
results can’t be compared to
each other because there are
too many parameters that
differ from each other. The
results of batch 1 are
described in the results
section. Results of batch 2
aren’t discussed. CREB
shows down regulation in
both UFP/Rest (R=0.661; P=
0.02) and Ambient
air/Exercise (R= 0.84; P=
0.001) groups compared with
the Ambient air/Rest group.
UCP2 (R= 0.864; R= 0.042)
is down regulated in the
ambient air/Rest group and
GAP43 (R= 0.849; P= 0.042)
shows decreased expression
in the UFP/Exercise group.
22
Appendix 8: Interplate calibrator
Table 2: The interplate calibrators show differences between different runs of the lightcycler 480. For example:
On the plate called SYT11, this primer was tested for each sample. In the empty wells, the household gene B2M
is retested in triplicate for one sample (CE24H3). On the next plate, UCP2 was tested for all samples. Again in
the remaining wells the same samples combined with the B2M primer was loaded in triplicate. These values may
not differ too much from each other in order to exclude interassay variations. In this study B2M and BDNF are
used as calibrators. The variance is calculated by the subtraction of the highest value and the lowest value of the
same primer combined with the same sample on different plates to see whether there are differences between
different runs. Variances did not reach too high values.
Interplate Calibrators
Place
Sample
Cp
primer
Plate
Average
Variance
B11
CE24H3
23
B2M
SYT11
0,1
C11
CE24H3
23,08
B2M
SYT11
D11
CE24H3
23,05
B2M
SYT11
B11
CE24H3
22,9
B2M
UCP2
C11
CE24H3
23,27
B2M
UCP2
D11
CE24H3
23,13
B2M
UCP2
B11
CE24H3
23,04
B2M
GAP43
C11
CE24H3
23,5
B2M
GAP43
D11
CE24H3
23,26
B2M
GAP43
B11
CE24H3
22,96
B2M
CAMK2a
C11
CE24H3
23,28
B2M
CAMK2a
D11
CE24H3
23,08
B2M
CAMK2a
E11
CE24H3
31,7
BDNF
SYT11
0,11
F11
CE24H3
31,67
BDNF
SYT11
G11
CE24H3
31,68
BDNF
SYT11
E11
CE24H3
31,67
BDNF
GAP43
F11
CE24H3
31,7
BDNF
GAP43
G11
CE24H3
31,73
BDNF
GAP43
E11
CE24H3
31,77
BDNF
CAMK2a
F11
CE24H3
31,59
BDNF
CAMK2a
G11
CE24H3
31,69
BDNF
CAMK2a
B12
CR24H5
22,6
B2M
SYT11
1,41
C12
CR24H5
22,59
B2M
SYT11
D12
CR24H5
22,52
B2M
SYT11
B12
CR24H5
22,53
B2M
UCP2
C12
CR24H5
21,76
B2M
UCP2
D12
CR24H5
21,19
B2M
UCP2
B12
CR24H5
22,69
B2M
GAP43
C12
CR24H5
22,97
B2M
GAP43
D12
CR24H5
22,54
B2M
GAP43
B12
CR24H5
22,68
B2M
CAMK2a
C12
CR24H5
22,65
B2M
CAMK2a
D12
CR24H5
22,66
B2M
CAMK2a
E12
CR24H5
31,62
BDNF
SYT11
0,14
F12
CR24H5
31,74
BDNF
SYT11
G12
CR24H5
31,48
BDNF
SYT11
E12
CR24H5
31,54
BDNF
GAP43
23
F12
CR24H5
31,81
BDNF
GAP43
G12
CR24H5
31,61
BDNF
GAP43
E12
CR24H5
31,5
BDNF
CAMK2a
F12
CR24H5
31,67
BDNF
CAMK2a
G12
CR24H5
31,49
BDNF
CAMK2a
B10
E7H
23,2
B2M
SYT11
1,26
C10
E7H
22,71
B2M
SYT11
D10
E7H
22,54
B2M
SYT11
B10
E7H
23,1
B2M
UCP2
C10
E7H
22,07
B2M
UCP2
D10
E7H
21,94
B2M
UCP2
B10
E7H
23,32
B2M
GAP43
C10
E7H
22,8
B2M
GAP43
D10
E7H
22,9
B2M
GAP43
B10
E7H
23,21
B2M
CAMK2a
C10
E7H
22,9
B2M
CAMK2a
D10
E7H
22,93
B2M
CAMK2a
E10
E7H
31,21
BDNF
SYT11
0,13
F10
E7H
31,26
BDNF
SYT11
G10
E7H
31,08
BDNF
SYT11
E10
E7H
31,45
BDNF
GAP43
F10
E7H
31,2
BDNF
GAP43
G10
E7H
31,57
BDNF
GAP43
E10
E7H
31,25
BDNF
CAMK2a
F10
E7H
31,32
BDNF
CAMK2a
G10
E7H
31,32
BDNF
CAMK2a
