Negative effects of ultrafine particle exposure during forced exercise on the expression of Brain-Derived Neurotrophic Factor in the hippocampus of rats

Neuroscience 223 (2012) 131–139

NEGATIVE EFFECTS OF ULTRAFINE PARTICLE EXPOSURE DURING FORCED EXERCISE ON THE EXPRESSION OF BRAIN-DERIVED NEUROTROPHIC FACTOR IN THE HIPPOCAMPUS OF RATS I. BOS, a,b P. DE BOEVER, a,c L. INT PANIS, a,d S. SARRE e AND R. MEEUSEN b*

cise-induced up-regulation of BDNF gene expression in the hippocampus of rats. Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

a

Environmental Risk and Health, Flemish Institute for Technological Research (VITO), Mol, Belgium

b

Human Physiology & Sports Medicine, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium

Key words: air pollution, particulate matter, exercise, BDNF, prefrontal cortex, olfactory bulb.

c

Centre for Environmental Sciences (CMK), Hasselt University, Agoralaan, Building D, 3590 Diepenbeek, Belgium d

Transportation Research Institute (IMOB), Hasselt University, Wetenschapspark 5, 3590 Diepenbeek, Belgium

INTRODUCTION

e

Pharmaceutical Chemistry, Drug Analysis and Drug Information, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium

Particulate matter (PM) is a component of air pollution. It comprises a mixture of solid particles and liquid droplets suspended in the air (Block and Calderon-Garciduenas, 2009). PM is broadly categorized based on aerodynamic diameter into particles <10 lm (PM10), particles <2.5 lm (PM2.5), and the ultrafine particles <100 nm (UFP) (Brook et al., 2010). Because of its small size, UFP may enter the body more easily than the larger particles and may be more potent to induce health effects (Seaton et al., 1995) even beyond the respiratory tract, in the central nervous system for example (Block and CalderonGarciduenas, 2009). Postmortem studies in healthy children and young adults have shown associations between long-term PM exposure and deposition of UFP in olfactory bulb neurons, neuroinflammation, disruption of blood–brain barrier, and accumulation of amyloid b42 and a-synuclein (Caldero´n-Garciduen˜as et al., 2008a, 2012). Furthermore, several authors report associations between living in a polluted environment with high PM concentrations and cognitive decline (Caldero´nGarciduen˜as et al., 2008b, 2011; Suglia et al., 2008; Chen and Schwartz, 2009; Ranft et al., 2009). Inflammation and oxidative stress are considered two basic mechanisms through which PM induces negative health effects, including adverse effects in the brain (Mohankumar et al., 2008; Block and Calderon-Garciduenas, 2009; Brook et al., 2010). Animal studies also confirmed effects of short-term exposure to PM. Campbell et al. (2005) found increased levels of inflammatory biomarkers IL1a, TNFa and NFkB in the brain of ovalbumin sensitized mice exposed to high levels of particles for 2 weeks. Tin-TinWin-Shwe et al. (2008) found that a single intranasal administration of Carbon Black increased IL1b mRNA synergistically with lipoteichoic acid in mice. Regular physical activity improves cardiovascular fitness, as well as cognitive function (Masley et al., 2009; Stroth et al., 2009) and psychological health (Antunes et al., 2005; Nabkasorn et al., 2006).

Abstract—Exercise improves cognitive function, and BrainDerived Neurotrophic Factor (BDNF) plays a key role in this process. We recently reported that particulate matter (PM) exposure negatively contributed to the exercise-induced increase in human serum BDNF concentration. Furthermore, PM exposure is associated with neuroinflammation and cognitive decline. The aim of this study was to investigate the effect of exposure to ultrafine particles (UFP) during a single bout of forced exercise on the expression of inflammatory (IL1a, IL1b, TNF, IL6, NOS2, NOS3) and oxidative stress (NFE2L2)-related genes, as well as BDNF in the brain of rats. Four groups (n = 6/group) of Wistar rats were exposed for 90 min to one of the following exposure regimes: UFP + exercise, UFP + rest, ambient air + exercise, ambient air + rest (control). Hippocampus, olfactory bulb and prefrontal cortex were collected 24 h after exposure. Gene expression changes were analyzed with real-time PCR. In the condition ambient air + exercise, hippocampal expression of BDNF and NFE2L2 was up-regulated, while the expression of IL1a and NOS3 in the prefrontal cortex and IL1a in the olfactory bulb was down-regulated compared to the control. In contrast, gene expression in the condition UFP + exercise did not differ from the control. In the condition UFP + rest, hippocampal expression of NFE2L2 was down-regulated and there was a trend toward down-regulation of BDNF expression compared to the control. This study shows a negative effect of UFP exposure on the exer*Corresponding author. Tel: +32-2-6292222; fax: +32-2-6292876. E-mail addresses: [email protected] (I. Bos), [email protected], [email protected] (R. Meeusen). Abbreviations: B2M, b-2 microglobulin; BDNF, Brain-Derived Neurotrophic Factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPRT1, hypoxanthine phosphoribosyltransferase 1; IL1a, interleukin 1 alpha; IL1b, interleukin 1 beta; IL6, interleukin 6; NFE2L2, nuclear factor, erythroid-derived 2, like 2; NOS2, nitric oxide synthase 2, inducible; NOS3, nitric oxide synthase 3, endothelial; PM, particulate matter; TNF, tumor necrosis factor; UFP, ultrafine particles; YWHAZ, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide.

0306-4522/12 $36.00 Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2012.07.057 131

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Also in rats, it was demonstrated that after a period of ‘‘voluntary’’ running in a running wheel or ‘‘forced’’ exercise on a treadmill learning and memory is improved (Vaynman et al., 2004; Ang et al., 2006; Aguiar et al., 2011). A training period as well as a single bout of forced exercise increases mRNA and protein levels of the neurotrophin, Brain-Derived Neurotrophic Factor (BDNF) in the hippocampus (Huang et al., 2006; Soya et al., 2007; Aguiar et al., 2011). The up-regulation of BDNF levels in the hippocampus seems to play a key role in the underlying process leading to improved learning and memory in response to exercise (Vaynman et al., 2004; Vaynman and Gomez-Pinilla, 2005). In view of the health benefits, many people are committed to a regular exercise program. However, they cannot always avoid exercising in close proximity to traffic resulting in exposure to traffic exhaust, a major source of UFP (Janhall et al., 2004). Recent findings suggest that personal exposure to the black carbon fraction of PM is related to traffic participation and associated with important health impacts (Jacobs et al., 2010; Dons et al., 2011; Janssen et al., 2011; von Klot et al., 2011). Additionally, exercise significantly increases exposure to PM (Int Panis et al., 2010). Considering the negative consequences of PM, it is not clear whether exercising in polluted air still brings the above mentioned benefits. In humans, peripheral BDNF levels increase acutely in response to an exercise bout and these increases are suggested to be reflected in the brain (Rojas Vega et al., 2006; Ferris et al., 2007; Rasmussen et al., 2009; Goekint et al., 2011; Griffin et al., 2011). Moreover, it is suggested that exercise increases serum BDNF levels in humans by increasing production and release from brain areas like the hippocampus (Rasmussen et al., 2009). However, we found that the exercise-induced increase in the level of serum BDNF was not present in humans 30 min after cycling near a busy road (Bos et al., 2011). We hypothesized that UFP may interfere with the increased production of BDNF in the brain. Since BDNF expression in the rat brain is increased in response to exercise, the aim of this paper is to investigate whether UFP exposure inhibits the exercise-induced up-regulation of BDNF expression in the brain of rats. In this study, we hypothesized that a short bout of forced exercise would increase hippocampal BDNF expression in rats. Furthermore, UFP exposure could affect the exerciseinduced increase of BDNF expression. Additionally, the acute effect of UFP exposure will be analyzed through the expression of genes related to inflammation (IL1a, IL1b, TNF, IL6, NOS2, NOS3) and oxidative stress (NFE2L2).

EXPERIMENTAL PROCEDURES Animals, animal training and study approval Twenty-four male, albino, Wistar rats, weighing 175–200 g, were purchased from Charles River Laboratories (Ko¨ln, Germany). Rats were housed in groups in a room on a 12-h light–dark cycle for 6 days before starting treadmill familiarization. Animals had a standardized diet with food and water ‘ad libitum’.

Protocols were in accordance with national rules on animal experiments and were approved by the Ethics Committee on Animal Experiments of the Faculty of Medicine and Pharmacy of the Vrije Universiteit Brussel (Brussels, Belgium). All efforts were made to minimize animal suffering and the minimum number of animals necessary to produce reliable scientific data was used. The animals were familiarized with treadmill running during the week before the experiment on a motor driven horizontal treadmill inside the exposure compartment (Omnitech electronics, Columbus, Ohio, USA) using mild electric shocks of 0.8 mA. The familiarization protocol consisted of short exercise bouts during 5 consecutive days. Treadmill speed and running duration were steadily increased each day until the rats were able to run 75 min at 20 m min1. Subsequently, they were randomly allocated to 4 groups of 6 animals each. The 4 groups corresponded to 4 different 90-min exposure regimes: (1) UFP-polluted air + EXERCISE, (2) UFP-polluted air + REST, (3) ambient air + EXERCISE, and (4) ambient air + REST (control). The rats that took part in the exercise protocol ran for 90 min at a speed of 20 m min1 (EXERCISE groups), the other rats were sitting still in the exposure compartment (REST groups). The rats were euthanized 24 h after the exposure with an overdose of natriumpentobarbital (Nembutal, Ceva Sante Animale, Brussels, Belgium) injected intraperitoneally. The brain was excised and three brain regions (hippocampus, prefrontal cortex and olfactory bulb) were dissected and stored overnight at 4 °C in RNAlater to stabilize the RNA (Life Technologies, Ghent, Belgium). Next, the RNAlater was removed by decantation and the samples were stored immediately at 80 °C.

Exposure set-up The mini Combustion Aerosol Standard (miniCAST) model 6203-A (Jing Ltd, Zollikofen, Switzerland) was used to create a peak UFP concentration in the exposure compartment. The miniCAST generates a combustion particle soot flow of 3 l min1 with a constant particle mass and number concentration. The soot particles from the CAST are agglomerates of spherical soot particles. The particles have morphology similar to soot particles generated by diesel engines (Zahoransky et al., 2003). The size of the particles generated by the miniCAST can be adjusted in the range from 10 to 160 nm (www.sootgenerator.com/ miniCAST_g.htm). The miniCAST was fueled with propane gas in our experiments and the raw exhaust was diluted with ambient air (35 l – max 50 l min1). The exposure compartment had Plexiglas walls and roof and contained a treadmill inside. Particle concentration and size distribution inside the exposure compartment was monitored in real-time with a DMS50 Fast Particulate Size Spectrometer (Cambustion Ltd., Cambridge, United Kingdom) measuring particles in a size range between 5 and 560 nm. The carbon dioxide (CO2) level inside the exposure compartment was monitored with SIDOR Extractive Gas Analyzer (SICK MAIHAK Inc., Meersburg, Germany).

Gene expression analysis Tissue maintenance, total RNA extraction. RNA was extracted from the brain samples with the RiboPure kit (Life Technologies) according to the manufacturer’s instructions. Tissue samples were weighed and homogenized in 10–20 volumes of TRI reagent using a rotor–stator homogenizer. Total RNA was extracted from the homogenate with glass-fiber filter purification methodology and eluted with a low salt buffer. The RNA concentration was determined using a NanoDrop Spectrophotometer (Isogen Life Science, De Meern, The Netherlands), and quality and integrity of the extracted RNA was tested on the Agilent 2100 BioAnalyzer with capillary gelelectrophoresis using RNA 6000 Nano Chips (Agilent Technologies, Diegem, Belgium). RNA was stored in elution solution (Life Technologies) at 80 °C until further use.

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I. Bos et al. / Neuroscience 223 (2012) 131–139 Table 1. Panel of genes analyzed together with the gene description Gene symbol

Description

Assay name

BDNF

Brain-Derived Neurothrophic Factor b-2 Microglobulin Glyceraldehyde-3-phosphate dehydrogenase Hypoxanthine phosphoribosyltransferase 1 Interleukin 1 alpha Interleukin 1 beta Interleukin 6 Nuclear factor, erythroidderived 2, like 2 Nitric oxide synthase 2, inducible Nitric oxide synthase 3, endothelial Tumor necrosis factor Tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase activation protein, zeta polypeptide.

Rn.PT.42.10133431

B2M GAPDH HPRT1 IL1a IL1b IL6 NFE2L2 NOS2 NOS3 TNF YWHAZ

Rn.PT.42.8396530 Rn.PT.42.12624405 Rn.PT.42.14024643 Rn.PT.42.10241273 Rn.PT.42.14101013 Rn.PT.42.9109300 Rn.PT.42.9129229 Rn.PT.42.13446725 Rn.PT.42.6917365 Rn.PT.42.11142874 Rn.PT.42.8368619

PrimeTimeTM assay used for quantifying gene expression is mentioned. B2M, GAPDH, HPRT1, and YWHAZ were considered as housekeeping genes; the other genes were genes of interest in the real-time PCR analysis.

Real-time PCR protocol. The synthesis of cDNA started from 1-lg total RNA using a combination of oligo (dT)18 primer and random hexamers. This was done with the Transcriptor First Strand cDNA Synthesis Kit following the instructions of the manufacturer (Roche, Vilvoorde, Belgium). All reverse transcription reactions were done in a Veriti 96-well Thermal Cycler of Applied Biosystems (Life Technologies). Real time PCR amplifications were in 20 lL aliquots in 96-well plates using a Roche LightCycler480. The reaction mixtures consisted of 5 lL cDNA sample and 15 lL reaction mix (10 lL LightCycler480 Probes Master Kit (Roche), 1 lL primer-probe mix, and 5 lL nuclease-free water). We have used 50 exonuclease assays (PrimeTimeTM assays) from Integrated DNA Technologies (Leuven, Belgium). Details of genes that were analyzed, and the PrimeTimeTM assays that were used are given in Table 1. The real-time PCR protocol consisted of 1 cycle of preincubation (10 min at 95 °C), 45 cycles of amplification (10 s at 95 °C, 30 s at 62 °C, and 1 s at 72 °C) and 1 final cycle (10 s at 40 °C). All analyses were run in triplicate. Real-time PCR analysis generated sample specific crossing points (Cp). In the LightCycler software the ‘‘Second Derivative Maximum Method’’ is performed where Cp is automatically identified and measured at the maximum acceleration of fluorescence (Rasmussen, 2001).

meanf½ðefficiencyÞAverage

Cp ðcontrolÞAverage Cp ðsampleÞ

reference

gene 1 ;

Average Cp ðcontrolÞAverage Cp ðsampleÞ

½ðefficiencyÞ reference gene 2 ; . . .g. The normalized, relative gene expression ratio was calculated for each of the three groups (UFP-polluted air + EXERCISE, UFPpolluted air + REST, and ambient air + EXERCISE) relative to the control group (ambient air + REST). Statistical significance of the calculated relative expression ratio is subsequently analyzed using Pair Wise Fixed Reallocation Randomization TestÓ (Pfaffl et al., 2002; Pfaffl, 2004) with 2000 permutations. PCR efficiency was considered 100% for the 50 -exonuclease assays. REST was used because of two advantages over other relative quantification strategies for real-time PCR data. REST implements a mathematical model that allows for relative quantification between groups in contrast to previous mathematical models that calculate relative expression ratios between two samples. Furthermore, REST has implemented a valid statistical test for relative gene expression analysis using the Pair Wise Fixed Reallocation Randomization TestÓ. Statistical tests to determine accuracy of relative expressions are complex because ratio distributions do not have a standard deviation. REST 2009 software overcomes this problem by using a simple statistical randomization test that has the advantage of making no assumptions about the distribution of the data, while remaining as powerful as parametric tests (Pfaffl et al., 2002; Pfaffl, 2004). The randomization test uses a P(H1) test for the statistical analysis that represents the probability of the alternate hypothesis that the difference between the sample and the control group is because of chance only. The hypothesis test performed in our study 2000 random reallocations of samples and controls between the 2 groups and counts the number of times the relative expression on the randomly assigned group is greater than the sample data.

RESULTS Exposure assessment Particle concentrations generated by miniCAST were continuously measured inside the exposure compartment. The size of the particles measured by the DMS50 inside the exposure compartment ranged between 5 and 115 nm, covering the size range of UFP. The mean total particle concentration for the EXERCISE AND REST group exposed to UFP-polluted air was 1.24  107 (SD = 1.7  106) particles cm3 and 1.15  107 (SD = 2.4  106) particles.cm3, respectively. Particle concentrations were not significantly different (t(8) = 0.700, p = 0.504). The CO2 level inside the exposure compartment ranged within 1000–2500 ppm during the UFP exposure.

Gene expression analysis Statistical analysis Mean total particle concentrations between the EXERCISE- and REST – groups that were exposed to UFP, were compared using independent samples T-test. Three technical replicates of the real-time PCR analysis were averaged. Stability of the putative housekeeping genes (B2M, GAPDH, HPRT1, YWHAZ) was verified using geNorm (Vandesompele et al., 2002). Statistical analysis was performed using the Relative Expression Software Tool (RESTÓ 2009 V.2.0.13; Pfaffl et al., 2002). REST calculates the normalized, relative gene expression ratio (R) between two treatment groups using the mathematical model: relative expression ratiotarget gene ¼ ½ðefficiencyÞAverage

Cp ðcontrolÞAverage Cp ðsampleÞ

target

gene =Geometric

Total RNA was extracted successfully from olfactory bulb, prefrontal cortex and hippocampus. The average total RNA concentration was 207 ± 66 ng/lL. The RNA quality assessed with spectrophotometry was high with an A260/A230 ratio of 1.81 ± 0.05 and an A260/A280 ratio of 2.05 ± 0.05. The RNA integrity number was 8.68 ± 0.37. There was no difference in RNA quantity and quality between the different tissues or exposure groups. Real-time PCR analysis was reliable with technical replicates varying not more than 0.5 Cp unit. The expression of all genes, except IL6, could be detected. The latter could only be detected at Cp more

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UFP/Rest

*

Ambient air/Exercise

*

Expression ratio

2

UFP/Exercise

1.5

1

0.5

0

* BDNF

IL1α

IL1β

NFE2L2

NOS2

NOS3

TNF

Fig. 1. Hippocampal gene expression of selected genes. Data are expressed relative to the control – group (ambient air/rest). Error bars indicate standard error, n = 6 for each treatment. Significance is indicated (⁄p < 0.05) for each represented condition relative to the control condition.

than 36, and was not taken into account for further analysis. Stability of the housekeeping genes (HPRT1, B2M, GAPDH, and YWHAZ) was evaluated for each tissue using gene expression stability (M) value. The original geNORM publication proposes to use a threshold of M 6 0.5 to select the most stable housekeeping genes (Vandesompele et al., 2002). In our analysis the M-value for the four genes was always 60.5 and all genes were used for normalization of the genes of interest. Calculation of the normalized, relative gene expression and statistical interference was done using RESTÓ 2009 software. The results of three groups (UFP-polluted air + EXERCISE, UFP-polluted air + REST, and ambient air + EXERCISE) were compared to the control group (ambient air + REST). In the hippocampus (Fig. 1), BDNF expression was significantly increased (R = 1.543, p = 0.034) as well as NFE2L2 expression (R = 1.409, p = 0.033) in the condition ambient air + EXERCISE compared to the

control group. In the same condition ambient air + EXERCISE, a down-regulation of IL1a expression (R = 0.774, p = 0.046) and NOS3 expression (R = 0.683, p = 0.047) was found in the prefrontal cortex (Fig. 2) as well as a down-regulation of IL1a expression (R = 0.598, p = 0.029) in the olfactory bulb (Fig. 3) compared to the control group. In contrast, in the condition UFP-polluted air + EXERCISE, there were no significant differences in gene expression compared to the control. In the condition UFP-polluted air + REST, hippocampal expression of NFE2L2 was significant down-regulated (R = 0.580, p = 0.017) and hippocampal BDNF expression showed a trend toward a down-regulation (R = 0.657, p = 0.059) compared to the control group.

DISCUSSION This study shows increased hippocampal BDNF gene expression in response to exercise in ambient air, while this exercise-induced increase disappeared when

2.5

UFP/Rest Ambient air/Exercise

Expression ratio

2

UFP/Exercise

1.5

1

0.5

* 0

BDNF

IL1α

* IL1β

NFE2L2

NOS2

NOS3

TNF

Fig. 2. Gene expression of selected genes in the prefrontal cortex. Data are expressed relative to the control – group (ambient air/rest). Error bars indicate standard error, n = 4–6 for each treatment. Significance is indicated (⁄p < 0.05) for each represented condition relative to the control condition.

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I. Bos et al. / Neuroscience 223 (2012) 131–139 2.5

UFP/Rest Ambient air/Exercise UFP/Exercise

Expression ratio

2

1.5

1

0.5

* 0

BDNF

IL1α

IL1β

NFE2L2

NOS2

NOS3

TNF

Fig. 3. Gene expression of selected genes in the olfactory bulb. Data are expressed relative to the control – group (ambient air/rest). Error bars indicate standard error, n = 5–6 for each treatment. Significance is indicated (⁄p < 0.05) for each represented condition relative to the control condition.

exercise is performed in UFP-polluted air. Previous animal research already demonstrated an increase in hippocampal BDNF mRNA and protein levels after a single bout of treadmill exercise (Huang et al., 2006; Soya et al., 2007). In our study, an increase in BDNF mRNA levels was found 24 h after the exercise bout, while others detected an increase within 1–4 h following a single bout of treadmill exercise (Huang et al., 2006; Soya et al., 2007). The up-regulation of BDNF mRNA expression is suggested to be dose-dependent for exercise duration (Adlard et al., 2004), while the effect of exercise intensity is less clear. We used a moderate intensity of 20 m/min which is in the same range of intensity as previous studies showing increased BDNF expression. However, our exercise duration of 90 min is longer than the 30 min of exercise previously used, suggestive for an enhanced effect (Huang et al., 2006; Soya et al., 2007). In contrast, the rats that were exposed to the identical exercise protocol in an environment with high UFP levels did not show an increase in BDNF expression 24 h after the exercise bout. Although we recognize that the exercise-induced BDNF up-regulation may still be present at earlier time-points such as 1–4 h after UFP exposure, our findings suggest that UFP exposure may interfere with the increased production of BDNF in the brain of rats in response to exercise. These observations are in line with our previous findings in humans, where the exercise-induced increase in serum BDNF protein level was not present after cycling near a busy road with high concentrations of PM10, PM2.5 and UFP (Bos et al., 2011). In humans, peripheral BDNF levels increase acutely in response to an exercise bout (Rojas Vega et al., 2006; Ferris et al., 2007; Goekint et al., 2011; Griffin et al., 2011). It is suggested that exercise increases serum BDNF levels in humans by increasing production and release of BDNF from brain areas like the hippocampus (Rasmussen et al., 2009). However, we found that the exercise-induced increase in the level

of serum BDNF was not present in humans 30 min after cycling near a busy road and hypothesized that PM exposure may interfere with the increased production of BDNF in the brain (Bos et al., 2011). The exerciseinduced increase in BDNF level is considered a key mechanism through which exercise improves cognition (Vaynman et al., 2004; Vaynman and Gomez-Pinilla, 2005). Our findings suggest that high UFP exposure during exercise along busy roadways and in urbanized environment could counteract the beneficial increase of BDNF levels, and may therefore compromise cognitive improvements. This is not the first study in which exercise fails to increase BDNF mRNA levels. Vaynman et al. (2006) found that hippocampal injection of a drug that disrupts mitochondrial metabolism and increases oxidative stress inhibits the exercise-induced increase in BDNF mRNA and protein. Also Griesbach et al. (2004a,b) demonstrated that voluntary exercise early after induction of mild traumatic brain injury (TBI) failed to increase hippocampal BDNF levels. They suggested that the dynamic neurochemical and metabolic alterations induced by TBI may interfere with the effects of exercise. The pathways through which UFP exposure might interact with BDNF expression and interferes with the effect of exercise may be linked with oxidative stress and inflammation. Chronic exposure to PM is associated with increased oxidative stress and proinflammatory events in the brain (Campbell et al., 2005, 2009; Caldero´n-Garciduen˜as et al., 2008b, 2012; Mohankumar et al., 2008). Oxidative stress and inflammation are considered as main mechanisms through which PM induces negative health effects (Block and Calderon-Garciduenas, 2009; Brook et al., 2010). Literature concerning the effect of oxidative stress on the BDNF levels suggests that increased oxidative stress that causes oxidative damage decreases BDNF expression (Wu et al., 2004; Vaynman et al., 2006). Also inflammation is linked to decreased BDNF expression (Barrientos et al., 2004; Cortese et al., 2011). Infusion of the inflammatory

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cytokine IL1b in the hippocampus decreases its capacity for BDNF expression (Barrientos et al., 2004). In our study, the expression of inflammatory and antioxidant defense genes was not significantly upregulated in response to acute UFP exposure. This is in contrast to studies on chronic exposure suggesting that the dose of this single, short lasting UFP exposure is too small to activate defensive gene expression (Campbell et al., 2005, 2009; Hartz et al., 2008; Levesque et al., 2011). Tin-Tin-Win-Shwe et al. (2008) did find increases in IL1b mRNA 11 h after a single intranasal administration of nano-sized Carbon Black to mice, but this was administered together with lipoteichoic acid which may potentiate the neurological effect of the nanoparticles. Another explanation might be that the time-course of the effect differs and that any increase in expression of inflammatory and antioxidant genes had already decreased after 24 h. We found a trend toward a downregulation of BDNF expression in response to UFP exposure. Previously, chronic cigarette smoke exposure was found to induce oxidative damage and decrease hippocampal BDNF levels in mice (Tuon et al., 2010). We suggest that increasing the duration of UFP exposure so that it augments oxidative stress and induces an inflammatory response, might lead to a significant downregulation of BDNF expression. In contrast to UFP, exercise has a beneficial impact on inflammation and oxidative stress in the brain (Vollert et al., 2011; Barrientos, 2011). This seems to be supported by our own findings. The group exercise/ambient air showed decreased expression of inflammatory markers, IL1a and NOS3 in the prefrontal cortex and Il1a in the OB. Although the few studies concerning the acute effect of exercise on inflammatory markers in the brain show increases (Packer et al., 2010), a decreased expression of inflammatory markers is in agreement with multiple studies on chronic exercise showing a diminished inflammation state in the brain of exercising animals (Barrientos, 2011; Funk et al., 2011; Leem et al., 2011). A decreased inflammation state is considered beneficial as chronic inflammation is involved in multiple neurodegenerative diseases (Barrientos, 2011). In addition, hippocampal expression of NFE2L2 was increased with exercise in ambient air. NFE2L2 is a redox-sensitive transcription factor that - upon activation by oxidative stress - activates expression of antioxidant genes. Upon activation of NFE2L2, the cellular protein content of NFE2L2 increases (Kwak et al., 2002; Nguyen et al., 2003, 2004). Although, single bouts of exercise do not cause oxidative damage or increased antioxidant status in the brain (Acikgoz et al., 2006; Aksu et al., 2009), exercise training induces an adaptive response that increases the brains capacity to cope with oxidative stress (Salim et al., 2010; Mazzola et al., 2011; Vollert et al., 2011). The finding that exercise increases NFE2L2 mRNA levels in the hippocampus, is in agreement with this adaptive process suggesting that exercise might improve its antioxidant defense system by stimulating the expression of the redox-sensitive transcription factor NFE2L2. In contrast, the rats that exercised in the UFPpolluted air did not have these beneficial effects on

expression of inflammatory and oxidative stress-related genes. This suggest that in addition to BDNF expression, also inflammatory and oxidative stress-related genes are regulated differently in response to the exercise in an environment with high UFP levels, although UFP exposure in the resting state did not significantly affect expression of these genes. However, PM exposure is known to increase during exercise due to increased ventilation, possibly increasing PM-related health effects (Jacobs et al., 2010). Thus, in addition to previous studies in which exercise fails to increase BDNF levels, this study provides further elements suggesting that UFP exposure during exercise might induce a different brain response on oxidative stress- and inflammation-related genes, counteracting the effect of exercise and leading to a lower level of BDNF expression. This study makes a valuable contribution to the research in the fact that it shows evidence that there is an increase in BDNF expression at 24 h after exercise. We argue, therefore, that the fact that the identical exercise protocol in the UFP-polluted air produces different effects on BDNF expression is convincing since we are able to compare two exposure regimes with identical exercise protocols. Future research should attempt to replicate the evidence shown here at different time points based on an accurate design which includes an experimental and a control group. We have chosen to perform analyses 24 h postexposure based on the study of Gerlofs-Nijland et al. (2005). They performed a time course analysis (4 h – 24 h – 48 h) of PM effects on the pulmonary and vascular system in spontaneously hypertensive rats and concluded that a 24-h interval from exposure to sacrifice seemed appropriate to assess the relative toxic potency of PM. In their study, the majority of the health effects were observed one day after PM exposure compared to the other times studied (Gerlofs-Nijland et al., 2005). It is possible that the exercise-induced BDNF upregulation is still present at earlier time-points, such as 1–4 h after UFP exposure. However, it was shown recently that gene expression in specific brain regions of rats was affected 18 h as well as 4 h after a 2-h diesel engine exhaust exposure (van Berlo et al., 2010). This indicates that the exercise-induced BDNF up-regulation may be affected also at earlier time-points. The contrast between both exposure environments was rather high because in the UFP condition, rats were exposed to high combustion particle concentrations that are only reached in extreme real world conditions (e.g. in tunnels). It was shown that the soot particles generated from miniCAST are very similar to soot particles from diesel exhaust (Zahoransky et al., 2003). Chemically the particles may be different because the miniCAST is fueled by propane gas and not diesel that contains trace amounts of sulfur and is normally mixed with metalcontaining additives. However, it is not clear whether it is the number, the physical size or certain chemical components that make PM harmful. As with traffic exposure, UFP exposure in our study was accompanied by noise. We do not exclude that noise, as a possible stress factor, could have confounded our results. Future

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challenges are to determine whether the noise that often accompanies PM exposure has an additive effect in the health effects of PM exposure. Additionally, changes in mRNA levels are not necessarily followed by the same changes in protein levels, so these findings need to be confirmed on protein level (Soya et al., 2007). Finally, by measuring the acute response at a single time-point 24 h following exposure, we might have missed earlier effects. A time-course experiment would give more insight into the actual timing and size of the response following UFP exposure. In this study, we analyzed the effect of UFP exposure during a ‘forced’ exercise bout using a motorized treadmill. Although forced exercise on a motorized treadmill was previously found to increase BDNF mRNA and protein levels and improve learning and memory (Ang et al., 2006; Huang et al., 2006; Soya et al., 2007; Aguiar et al., 2011), it differs from voluntary exercise in that it may increase stress (Huang et al., 2006). Since, humans normally engage in voluntary exercise, a protocol examining the effects of PM exposure during voluntary exercise may be more relevant for extrapolation to the human condition.

CONCLUSIONS Rats that ran for 90 min on a treadmill had increased hippocampal BDNF gene expression 24 h after the exercise bout. In contrast, rats submitted to the same exercise regime in an environment with a high UFP concentration did not have increased BDNF gene expression 24 h after the exercise bout. In addition, inflammation and oxidative stress-related gene expression was affected differently in the two exercise groups. Acknowledgements—Inge Bos was supported by a VITO PhDfellowship. We thank Johan Bruyninx and Rob Brabers for their technical help with the set-up of the UFP exposure compartment and Karen Hollanders for her excellent technical skills during the Q-PCR analyses.

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(Accepted 26 July 2012) (Available online 4 August 2012)