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Differential brain and spinal cord cytokine and BDNF levels in

experimental autoimmune encephalomyelitis are modulated by prior

and regular exercise

Danielle Bernardes

a,b

, Onésia Cristina Oliveira-Lima

a

, Thiago Vitarelli da Silva

a

, Camila Cristina Fraga Faraco

a,c

,

Hércules Ribeiro Leite

a

, Maria Aparecida Juliano

d

, Daniel Moreira dos Santos

e

, John R. Bethea

f

,

Roberta Brambilla

f

, Jacqueline M. Orian

g

, Rosa Maria Esteves Arantes

c

, Juliana Carvalho-Tavares

a,

aNúcleo de Neurociências, Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil bPrograma de Pós Graduação em Ciências Biológicas: Fisiologia-Farmacologia, CAPES Foundation, Ministry of Education of Brazil, Brasília, DF 70040-020, Brazil

cNeuroimunopatologia Experimental, Departamento de Patologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil dDepartamento de Biofísica, Universidade Federal de São Paulo, São Paulo, SP, 04023-062, Brazil

eLaboratório de Venenos e Toxinas Animais, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil fThe Miami Project To Cure Paralysis, Miller School of Medicine, University of Miami, 1095 NW 14th Terrace, Miami, FL 33136, USA

gDepartment of Biochemistry, La Trobe University, Bundoora, Victoria, 3086, Australia

a b s t r a c t

a r t i c l e

i n f o

Article history:

Received 5 June 2013

Received in revised form 14 August 2013 Accepted 26 August 2013

Keywords:

Experimental autoimmune encephalomyelitis Exercise

BDNF Neuroprotection Neuroinflammation Intravital microscopy

The interactions between a prior program of regular exercise and the development of experimental autoimmune encephalomyelitis (EAE)-mediated responses were evaluated. In the exercised EAE mice, although there was no effect on infiltrated cells, the cytokine and derived neurotrophic factor (BDNF) levels were altered, and the clinical score was attenuated. Although, the cytokine levels were decreased in the brain and increased in the spinal cord, BDNF was elevated in both compartments with a tendency of lesser demyelization volume in the spinal cord of the exercised EAE group compared with the unexercised.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Multiple sclerosis (MS) is a central nervous system (CNS) disease characterized by multifocal inflammatory infiltrates that are associated with the degradation of myelin and neuronal damage (Popescu and

Lucchinetti, 2012). These processes result in severe neurological

deficits, which profoundly affect the quality of life (Sung et al., 2013). However, because of the link between neurodegeneration and the inflammatory response, neuroprotection in MS has been described as a complex phenomenon (De Santi et al., 2011). The inflammatory response exacerbates the neuropathology but also stimulates the mech-anisms of tissue repair by secreting soluble factors, such as BDNF (brain-derived neurotrophic factor), which contributes to the controversy (Luhder et al., 2013). BDNF is an important molecular mediator of

neuronal survival (De Santi et al., 2011; Luhder et al., 2013) and has been associated with the beneficial effects of exercise on the CNS (Ang and Gomez-Pinilla, 2007; Basso and Hansen, 2011).

Because of the growing interest in the relationship between health-promoting behaviors and disease progression, the effect of physical exercise on MS has received considerable attention; however, the results of these studies are controversial (Motl and Pilutti, 2012). Additionally, the effects of exercise on MS pathogenesis have been addressed through analyses of cytokines and neurotrophic factors, which have generated inconclusive data. Indeed, previous reports have demonstrated that following participation in a regular exercise program serum pro-inflammatory cytokine levels in MS patients are unaltered (Heesen et al., 2003; Schulz et al., 2004; Bansi et al., 2012), decreased (Golzari et al., 2010) and increased (Castellano et al., 2008). Following a single exercise session (Gold et al., 2003) or a short period of exercise lasting 3 to 4 weeks (Castellano and White, 2008; Bansi et al., 2012), BDNF levels are increased; however, this increase is not observed after prolonged periods of exercise (Schulz et al., 2004; Castellano and White, 2008; Waschbisch et al., 2012).

Notably, higher serum levels of BDNF have been observed during MS relapse compared to the stable phase of the disease (Sarichielli et al., ⁎ Corresponding author at: Núcleo de Neurociências, Departamento de Fisiologia e

Biofísica, Instituto de Ciências Biológicas (ICB),Universidade Federal de Minas Gerais, Av Antônio Carlos 6627,Belo Horizonte, MG, 31270-901, Brazil. Tel.: +55 31 3409 2943; fax +55 31 3409 2924.

E-mail address:julianat@icb.ufmg.br(J. Carvalho-Tavares).

0165-5728/$–see front matter © 2013 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.jneuroim.2013.08.014

Contents lists available atScienceDirect

Journal of Neuroimmunology

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2002) and are inversely correlated with the number of T2 MRI lesions (Comini-Frota et al., 2012). These data suggest that strategies to improve BDNF release during relapse promote the addictive neuro-protective effect. However, for ethical purposes, only patients consid-ered clinically stable are included in studies analyzing the effects of exercise on MS, making it difficult to determine whether exercise pro-motes increased levels of BDNF during the acute phase of MS.

To further understand molecular regulation during the acute phase of the disease, mouse models of experimental autoimmune encephalo-myelitis (EAE) have been used. These models exhibit the characteristic clinically progressive caudo-rostral paralysis associated with the histo-logical hallmarks of the disease, including CNS inflammatory infiltrates, demyelination and axonal damage (dos Santos et al., 2005; Recks et al., 2011; Petratos et al., 2012). In rat and mouse EAE models, exercise has been demonstrated to have no effect (Patel and White, 2013) or to promote delayed (Le Page et al., 1994, 1996) or attenuated (Rossi et al., 2009) clinical score responses. However, it remains unclear if it is the exercise itself or the stress related to the exercise that contributes to the beneficial effects. To address this concern, long-term exercise (more than 3 weeks, which promotes the adaptation of the body to an attenuated stress response) has been used (Sigwalt et al., 2011). In this regard, a systematic protocol of regular exercise administered prior to the induction of and maintained until just before the develop-ment of the disease may be a useful strategy to investigate the interac-tions between regular physical exercise and disease neuroimmune modulatory responses during acute disease.

Swimming has been accepted as a well-controlled paradigm of physical training exercise. Swimming performed for 6 weeks has been shown to improve cardiovascularfitness (Evangelista et al., 2003), develop muscular endurance (Oh-ishi et al., 1996) and promote anti-inflammatory (Almeida et al., 2009) and neuroprotective effects (Deforges et al., 2009) in mice. Indeed, when compared to running, swimming may provide a more significant driving force for molecular adaptations in favor of neuroprotection (Ra et al., 2002; Deforges et al., 2009). Similarly, exercising when immersed in water is more effective at activating serum BDNF release in stable MS patients compared to training on land (Bansi et al., 2012). Therefore, investigat-ing the interactions between regular swimminvestigat-ing exercise and the neuroinflammatory responses of EAE during the acute phase of the disease may provide important information regarding the biological basis of MS progression and management of the disease.

In this study, we further investigated the effect of 6 weeks of prior swimming exercise training on the dynamics of inflammatory leukocyte recruitment, the lesion size and demyelination process, and the modu-lation of cytokines and BDNF, molecules associated with EAE induction.

2. Materials and methods

2.1. Animals and design

The Animal Care Facility at the Federal University of Minas Gerais (UFMG, Brazil) supplied the female C57BL/6 mice (6–8 weeks old). The animals were maintained on a 12:12 h light:dark cycle and were provided with food and water ad libitum. The mice were assigned to 2 groups: exercised and unexercised. Efforts were made to avoid any unnecessary distress to the animals, which was in accordance with NIH guidelines for the care and use of animals. The Institutional Animal Care and Use Committee (CETEA protocol number 24/2010) approved the protocols.

2.2. Physical exercise training protocol

The exercise training protocol was performed in a swimming pool, which was maintained at a controlled temperature (31±1 °C), for 30 min/day, 5 days/week, for 6 weeks. In thefirst week, the animals were subjected to progressive adaptation in the swimming pool (days

1 to 4) and a progressive load test (day 5), which consisted of an increasing workload corresponding to 2% of their body weight added every 3 min until exhaustion. The intensity of the exercise in the next endurance training session was set at 60% of the maximal weight obtained in the progressive load test. The maximal weight carried by the animal in the progressive load test was converted to a percentage of the animal's body weight. The mice were weighed weekly, and a new workload was determined using the previously calculated percent-age value. The test and workload adjustments were described previously (Almeida et al., 2009). The average workload was set up with an average of 7% of the animal's body weight. To account for the stress associated with the water environment, the unexercised mice were placed on a

flat open surface inside the swimming pool for the same length of time as the exercised group. To reduce the stress associated with the activa-tion of the body temperature control mechanism after each swimming exercise session, the animals from both groups were gently dried using a towel.

2.3. EAE induction and clinical assessment

The MOG35–55 peptide or saline injections (control group) were performed at the beginning of thefifth week of the physical exercise training protocol and the injections were continued until day 10 post-induction (10 dpi). The analyses were performed at 4–6 h after the last exercise session (10 dpi) or at 96 h (14 dpi). For IL-6, it has been suggested that 2–3 h of post-exercise time are required to return to baseline levels following a single bout of moderate-intensity exercise (Castellano et al., 2008). The control animals were analyzed 14 days after thefirst saline injection. EAE was induced as described previously (dos Santos et al., 2005, 2008) by subcutaneous immunization (in the tail base) with an emulsion containing 100μg of MOG35–55peptide in complete Freund's adjuvant (CFA), supplemented with 4 mg/ml

Mycobacterium tuberculosisH37Ra (Difco Laboratories, Detroit, MI, USA).

Bordetella pertussis toxin (300 ng/animal; Sigma-Aldrich, St. Louis, MO, USA) was injected i.p. on the day of immunization and after 48 h. The control animals received saline injections at similar times. Animal weight and clinical score were monitored daily as previously described (Kerfoot and Kubes, 2002; dos Santos et al., 2005, 2008). The scores were defined as follows: 0 = no clinical signs, 1 = tail paralysis (or loss of tail tone), 2 = tail paralysis and hind-limb weakness, 3 = hind-limb paralysis and 4 = complete hind-limb paralysis and front-limb weakness. This protocol promoted the development of EAE clinical signs, which were initially observed at 9–10 dpi and peaked at 13–14 dpi.

2.4. Intravital microscopy

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2.95 mol/L KCl, 1.71 mol/L CaCl2, 0.64 mol/L MgCl2, 24.6 mol/L NaHCO3, 3.71 mol/L dextrose and 6.7 mol/L urea; pH 7.4) at 37 °C. To assess the leukocyte–endothelium interactions, thefluorescent leuko-cytes were visualized under a Zeiss Imager M.2 (20x long-distance objective lens; Göttingen, Germany) equipped with afluorescent light source (epi-illumination at 510–560 nm, using a 590 nm emission

filter). Rolling leukocytes were defined as white cells moving at a veloc-ity less than that of erythrocytes and were expressed as the number. Leukocytes were considered adherent to the venular endothelium if they remained stationary for 20 s, which was the duration of the analy-sis. Microvessels were analyzed in sections 100μm in length, and the diameters of the vessels ranged from 40 to 115μm in the brain and 20 to 60μm in the spinal cord.

2.5. Histopathology

Animals were perfused (5 to 6 mice in each group) intracardially using 10% formalin in PBS and the tissues (brain and spinal cord) were maintained in thefixative until they were prepared for paraffin embed-ding. Brains were sectioned in the coronal plan at the level of the third and lateral ventricles. Spinal cords were sectioned transversally, and the thoracic-lumbar level was analyzed (the same region used for intra-vital microscopy). Serial sections (6μm) were cut and were stained using hematoxylin and eosin (HE). Spinal cord sections were additionally stained using Luxol Fast Blue (LFB) in order to evaluate demyelination levels. The images were visualized under an Olympus BX51 microscope (Tokyo, Japan) using 4×, 10× and 20× objective lenses. The images were captured at a resolution of 1392 × 1040 pixels using a digital camera coupled with image capture software (Pro-Express version 4.1 from Megacybernetics, Rockville, MD, USA) and a cool-snap kit. The HE images were analyzed qualitatively for the presence or absence of the inflammatory infiltrate.

2.6. Quantification of images

Spinal cord images stained with HE and LFB were quantified through optical density (OD) using the free Java image processing software

ImageJ(Image Processing and Analysis in Java) (http://rsbweb.nih.gov/ ij/). For this purpose, a representative spinal cord slice was collected from each animal in accordance with the above specifications using a 4× objective in 4 symmetrical quadrants (2 from the anterior funiculum and 2 from the posterior funiculum). Next, the images were converted into 8 bits and processed for low background through the automatic adjustment of the threshold using maximum entropy. The cellularity and myelin volume were analyzed as the equivalent of the pixel inten-sity in the HE and LFB stained sections, respectively.

2.7. Quantitative analysis of lesion size and demyelination burden

Paraffin embedding spinal cord tissues from 14 dpi animals (5 from exercised and 5 from unexercised groups) were additionally investigated to lesion size and demyelination burden. For this purpose, transverse sections (10μm) from thoracic-lumbar region were cut using a micro-tome (Leica, Germany) in a 1:10 series and mounted onto silame-coated glass slides. To quantify the area and volume of demyelinated white matter, two sequences offive serial sections with an equal distance (100μm) from each animal were stained with Luxol Fast Blue (counter-stained with HE). Stained sections were analysed using the Cavalieri's Principle with Stereo investigator software (Micro Bright Field, Inc.).

2.8. Measurement of cytokines

EAE mice (exercised and unexercised) and control mice (exercised and unexercised) were decapitated at 10 or 14 dpi (7 to 8 in each group). The brain and spinal cord were collected and stored in liquid nitrogen. Next, the tissues were homogenized in extraction solution

(100 mg/mL) containing 0.4 M NaCl, 0.05% Tween 20, 0.5% bovine serum albumin (BSA), 0.1 mM phenylmethylsulfonylfluoride, 0.1 mM benzetonium chloride, 10 mM EDTA and 20 KIU aprotinin prepared in phosphate-buffered saline (PBS). The homogenates were centrifuged at 10,000gfor 10 min at 4 °C, and the supernatants were stored at −20 °C. The IL-1β, IL-6, TNF, IL-10 and BDNF concentrations in the brain and spinal cord were determined at a 1:3 dilution in PBS contain-ing 0.1% BSA, uscontain-ing an ELISA kit in accordance with the manufacturer's instructions (R&D Systems, Minneapolis, MN and Pharmingen, San Diego, CA, USA). The concentration of each cytokine was calculated using a standard curve and was expressed as pg/mL.

2.9. Blood leukocyte count in the periphery

Before decapitation, 5μL of blood was collected from the tail of each mouse and diluted in 195μL of Turk solution (Merck, Darmstadt, Germany). The number of blood leukocytes in 10μL of the suspension was counted under light microscopy using a 10× objective lens (Olympus, Tokyo, Japan), and thefinal value was corrected for the dilu-tion factor. The blood leukocytes were collected from the control groups at 10 and 14 days post-saline injection, and the mice were decapitated at day 14 for the brain and spinal cord analyses.

2.10. Statistical analysis

Leukocyte recruitment was expressed as the median and range (Fig. 3); all other data were expressed as the mean ± SEM. The interac-tions between exercise training and the EAE model werefirst investi-gated using two-way ANOVA with the Bonferroni post-test. Multiple comparisons between the controls and the 2 EAE time points were performed using one-way ANOVA followed by the Tukey (cytokines

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and BDNF) or Kruskal–Wallis test, followed by the Dunn or Mann–

Whitney test (remaining data). Lesion size and myelin damage data from exercised and unexercised mice were analyzed by Studentttest. The significance level was established at Pb0.05.

3. Results

3.1. Swimming exercise for 6 weeks prior to disease development attenuates the severity of EAE from onset to disease peak

To assess the effect of prior physical exercise on the development of the clinical signs of EAE, the exercised (n = 33) and unexercised (n = 34) animals injected with MOG35–55were monitored from the day of induction until the disease peak (at 14 dpi), as previously described (dos Santos et al., 2005). Exercise training was initiated 4 weeks before induction and was maintained until 10 dpi, which corresponded to the timing of the onset of the clinical signs. Following the exercise-training protocol, we observed that the unexercised EAE animals exhibited a gradual increase in the clinical signs of the disease concomitant with the reduction in body mass; however, the exercised EAE animals demonstrated attenuation of the disease severity and body weight loss (Fig. 1). The clinical score (Fig. 1A) was associated with the number of days post-induction (F13,910= 147.73; Pb0.0001), exercise (F1,910= 42.92; Pb0.0001) and the interaction between these factors

(F13,910= 5.29; Pb0.0001). Significant differences were observed from 10 to 14 dpi (Pb0.05) and were confirmed by the Mann–

Whitney test (Pb0.05 at each time point).

For the relative weight (Fig. 1B), the comparison between both EAE groups also demonstrated an effect related to the dpi (F14,921= 43.02;

Pb0.0001), exercise (F1,921= 28,82; Pb0.0001) and the interaction

between both factors (F14,921= 3.29; Pb0.0001), with differences demonstrated at 10 to 14 dpi (Pb0.05). The comparison between

the 2 unexercised groups (control and EAE) demonstrated an effect as-sociated with dpi (F14,896= 14.66; Pb0.0001), EAE (F1,896= 448.41; Pb0.0001) and the interaction between these factors (F14,896= 28.93; Pb0.0001); significant differences (not shown) were observed

at 1 dpi (Pb0.05) and from 9 to 14 dpi (Pb0.001). The comparison

between the exercised groups (control and EAE) demonstrated an effect dependent on dpi (F14,879= 7.98; Pb0.0001), EAE (F1,879= 300.12; Pb0.0001) and the interaction between these factors (F14, 879= 17.35; Pb0.0001); significant differences (not shown) were observed at 1 dpi (Pb0.05), 3 dpi (Pb0.05) and 10 to 14 dpi (Pb0.001).

Nota-bly, for EAE induction, the 4 weeks of physical exercise training did not have a negative effect on body weight; the unexercised mice reached a weight of 18.93 ± 0.15 g (relative weight of 116.11 ± 0.89%), and the exercised animals reached a weight of 18.88 ± 0.16 g (relative weight 115.20 ± 0.80%; PN0.05 by unpairedt-test).

3.2. Swimming exercise for 6 weeks prior to disease development does not alter leukocyte recruitment to the CNS from onset to clinical peak in EAE mice

To understand the cellular mechanisms underlying the improve-ment in the clinical course of the disease over the 10 to 14 dpi period, in vivo leukocyte rolling and adhesion to the CNS vasculature were assessed (Figs. 2 and 3), followed by the evaluation of lesion develop-ment in the control and EAE groups (Fig. 4).

Intravital microscopy was used because this approach allows the quantitative evaluation of the dynamics of single-labeled cells in real

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time. We used the brain and spinal cord simultaneous windows tech-nique, which enabled us to visualize the microcirculation in both CNS compartments in the same animal using intravitalfluorescence. In the brain window, we observed a collecting major venule in a transverse position with a lateral to medial orientation that drained the blood from the brain postcapillary venules, and we collected data from 85.8 ± 7.2 vessels per group (10.5 ± 1.9 vessels/animal; F5,43= 1.67; PN0.05; one-way ANOVA for comparison between groups). In the spi-nal cord window, we also observed a collecting major venule at a central position, which collected blood from the spinal cord postcapillary venules, and we collected data from 79.7 ± 14.3 vessels per group (12.0 ± 2.5 vessels/animal; F5,34= 1.409; PN0.05; one-way ANOVA for comparison between groups). Animals that presented surgical trauma following the windows procedure were excluded from the experiments. The rolling and adhesion data were analyzed using the Kruskal–Wallis test (Pb0.0001) with Dunn's multiple comparison

post-test. In the control animals (exercised and unexercised), only 1–2 rolling or adherent cells perfield were observed in both the brain and spinal cord, and a significant difference was observed compared with the EAE groups (Pb0.05;Fig. 2A–D). However, no significant difference

in rolling or adhesion was observed between the exercised and unexer-cised groups in both the brain (Fig. 2A–B) and spinal cord (Fig. 2C–D) at either 10 or 14 dpi.

To our knowledge, this report is thefirst to analyze brain and spinal cord leukocyte recruitment in vivo in parallel in EAE mice. Because there were no differences between the exercised and unexercised groups and a similar response pattern was observed from 10 to 14 dpi, we

investigated the association between the clinical score and the recruit-ment of leukocytes in CNS compartrecruit-ments, brain and spinal cord. Addi-tional unexercised EAE mice analyzed between 10 and 14 dpi were included in this experiment to increase the degrees of freedom in each score. Next, the data were divided between scores ranging from 0 to 3–4, and we compared the data from the brain and spinal cord groups using the Mann–Whitney test. Interestingly, we observed that whereas the cerebral leukocyte rolling was decreased in animals with the highest clinical scores (2–4), it was not altered in the spinal cord (Fig. 3A). Cerebral leukocyte rolling in animals exhibiting scores between 0 and 1 was higher compared to that in animals exhibiting scores of 2 to 4. Additionally, spinal cord leukocyte rolling was higher than cerebral leukocyte rolling at scores between 2 and 4. Similarly, the spinal cord exhibited greater numbers of adherent leukocytes than the brain at scores between 3 and 4; however, the opposite was observed in animals with a score of 0 (Fig. 3B). Finally, using the diameter and length of the microvessels, we expressed the adhesion data as cells per mm2. The analysis corroborated the lack of differences between the exercised and unexercised animals in both the brain (Fig. 3C) and spinal cord (Fig. 3D). Additionally, this analysis demonstrated that the increased recruitment of cells in the spinal cord correlated with the increase in the clinical score (Fig. 3E).

To investigate the immunopathological basis of disease amelioration in the exercised EAE mice, lesion development was evaluated by histo-pathological analysis in the control and EAE groups. At 10 dpi, no evidence of cellular infiltration in either the brain or spinal cord in the EAE mice was observed in HE-stained tissues (data not shown), which Fig. 3.Comparison of leukocyte–endothelial interactions between the brain and spinal cord in EAE mice. Forfigures A, B and E, the data from 10 to 14 days post induction of EAE mice were distributed by clinical score (with 22, 07, 15 and 14 animals, respectively, in each range). Cerebral rolling and adhesion are represented by the white bars and symbols, and spinal cord rolling and adhesion are represented by the black bars and symbols. C is related toFig. 2B, D is related toFig. 2D, and E is related to Fig. 3B (adhesion is expressed as mm2). The data

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was consistent with the observation of a median clinical score of 0–1, indicative of symptoms restricted to the tail region. However, by 14 dpi, inflammation (meningeal and parenchymal) was pronounced in both the brain and spinal cord of the exercised and unexercised EAE mice (Fig. 4A–D). Therefore, a sharp increase in the cellularity (spinal cord sections only) was observed in EAE groups at 14 dpi (Fig. 4E); no significant difference was observed between the exercised and unexer-cised EAE groups. Moreover, a similar response was observed in the exercised and unexercised EAE groups for circulating leukocyte num-bers (Fig. 4F). Taken together, our data demonstrated that prior exercise did not affect the ability of the EAE animals to trigger an immune response in the periphery or cellular migration to the CNS parenchyma.

3.3. Swimming exercise for 6 weeks prior to disease development alters cytokine concentrations in CNS homogenates from onset to clinical peak in EAE mice

To investigate the role of soluble inflammatory mediators in the exercise-mediated amelioration of the disease, the cytokine concentra-tion was evaluated in non-perfused CNS homogenates from the exercised and unexercised mice (EAE and control groups). This analysis is relevant because cytokines play important roles in the polarization of immune responses. The panel of cytokines investigated included TNF and IL-1β (pro-inflammatory), IL-6 (inflammation-regulatory) and IL-10 (anti-inflammatory) (Figs. 5 and 6).

In the unexercised groups (control and EAE), a similar significant increase in cytokine levels at 10 dpi was observed in the brains of the unexercised EAE mice relative to the unexercised control animals (Fig. 5A–D). By 14 dpi, a reduction in the TNF, IL-1βand IL-10 levels (Fig. 5A, C and D, respectively) was observed in these animals, which

paralleled the numbers of circulating leukocytes (Fig. 4F). Comparison of the exercised and unexercised EAE groups at 10 dpi demonstrated that the TNF, IL-1βand IL-10 levels were significantly higher in the un-exercised group. For IL-6, no reduction was observed in the unun-exercised group between 10 and 14 dpi (Fig. 5B). In contrast, a significant differ-ence between the exercised EAE group and the unexercised EAE group was observed at 14 dpi (Fig. 5B). Given the significant decrease in the levels of blood leukocytes between 10 and 14 dpi, the data suggested an additional CNS contribution to the IL-6 levels.

Similar to the observations in the brain, in the spinal cord, the unex-ercised EAE group demonstrated increased IL-6 (Fig. 6B) and IL-1β (Fig. 6C) levels at 10 dpi related to the control; however, in the spinal cord, the levels of these cytokines were decreased at 14 dpi. Moreover, decreased TNF (Fig. 6A) and IL-10 (Fig. 6D) levels were observed at 14 dpi. The significant difference in the cytokine levels observed between 10 and 14 dpi corresponded to the circulating leukocyte numbers for the unexercised EAE mice (Fig. 4F). However, the exercised EAE mice exhibited a progressive augmentation in the TNF (Fig. 6A) and IL-6 (Fig. 6B) concentrations from 10 to 14 dpi. The exercised EAE mice exhibited significantly higher TNF, IL-6 and IL-10 concentrations at 14 dpi compared with their unexercised counterparts (Fig. 6A, B and D, respectively). This observation, combined with the absence of addi-tional circulating leukocytes (Fig. 4F) and the spinal cord recruitment of leukocytes (Figs. 2C, D,3D and4E), suggested a specific effect of prior exercise in modulating the production of these cytokines.

Interestingly, the exercised control animals demonstrated lower TNF concentrations in the brain (Fig. 5A) and spinal cord (Fig. 6A; Pb0.05

by unpairedttest) compared to the unexercised controls, suggesting a potential anti-inflammatory effect of this type of exercise under healthy conditions. This response was observed even with the increased Fig. 4.Histopathological analysis of exercised and unexercised EAE mice. Images of HE-stained brain (A, B) and spinal cord (C, D) sections at 14 dpi (n = 5–6 mice/group). The infiltration of mononuclear cells in the interventricular foramina (foramina of Monro) is visible in both the unexercised (A) and exercised (B) EAE mice and perivascular infiltration of the spinal cord in both the unexercised (C) and exercised (D) EAE mice. E) Optical density quantification of a single HE-stained spinal cord section from each mouse. Differences were observed between 14 and 10 dpi and between 14 dpi and the control in both the exercised and unexercised mice (Pb0.05). F) Blood leukocyte count in the periphery (n = 6–8 mice/group). Differences were observed between the control and EAE mice at 10 dpi (Pb0.01), between 10 and 14 dpi in the EAE mice (Pb0.05) and between the both time points for the exercised control group

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number of circulating leukocytes from 10 to 14 days post-saline injec-tion in the exercised control group (Fig. 4F; Pb0.05 by Mann–

Whitney test).

3.4. Swimming exercise for 6 weeks prior to disease development increases CNS homogenate BDNF concentrations at the clinical peak of EAE mice

BDNF has been associated with the beneficial effects of exercise on the CNS (Ang and Gomez-Pinilla, 2007; Basso and Hansen, 2011), with an improvement in EAE clinical symptoms (De Santi et al., 2009), and it is an important molecular mediator of neuronal survival (De Santi et al., 2011; Luhder et al., 2013). Therefore, we investigated the possible neuroprotective basis of disease amelioration in the exercised EAE mice by analyzing BDNF levels in brain and spinal cord homogenates.

Compared with unexercised control mice, the unexercised EAE mice demonstrated a significant increase in the BDNF concentration in the brain at 10 dpi (Fig. 7A), followed by a significant reduction by 14 dpi. However, a significantly higher BDNF concentration was observed at 14 dpi in the exercised EAE group compared with the controls and the exercised group at 10 dpi. These results demonstrated a significant difference in BDNF production between the exercised and unexercised EAE mice at both 10 and 14 dpi. In the spinal cord (Fig. 7B); compared with the control unexercised group, the unexercised EAE mice demon-strated a significant increase in the BDNF concentration at 10 dpi, which persisted at 14 dpi. The exercised mice only demonstrated a significant increase at 14 dpi compared to their 10 dpi and control counterparts. However, a significantly higher concentration of BDNF was also observed in the spinal cord of the exercised control mice compared to the unexercised control groups. In general, the exercised EAE mice

exhibited higher levels of BDNF production than the unexercised EAE mice in both the brain and spinal cord at 14 dpi.

3.5. Swimming exercise for 6 weeks prior to disease development slightly protected EAE mice from progression of demyelination from onset to clinical peak

To investigate the pathological basis of disease amelioration in the exercised EAE mice, demyelination spinal cord development was inves-tigated in the control and EAE groups. At 10 dpi, no evidence of demye-lination lesions in the EAE mice was observed in LFB-stained tissues (data not shown), which was consistent with the observation of symp-toms restricted to the tail region. By 14 dpi, demyelination lesions were evident in both exercised and unexercised EAE mice (Fig. 8A–B). How-ever, in contrast to exercised group, the progression of demyelination from 10 to 14 dpi was statistically different and more pronounced in the unexercised EAE group (Fig. 8C). At 14 dpi, the analysis of lesion size showed a value of 0.50 ± 0.08μm2× 106 for the unexercised group and 0.30 ± 0.10μm2× 106for the exercised group (difference of 0.20 ± 0.13; PN0.05 by unpairedt-test). Moreover, the analysis of

demyelination burden at 14 dpi by Cavalieri's Principle revealed values of 21.4 ± 4.3% for the unexercised group and 14.8 ± 4.2% for the exercised group (difference of 6.3 ± 6.0; PN0.05 by unpairedt-test).

4. Discussion

This study investigated the neuroinflammatory mechanisms under-lying the benefits of exercise training in murine EAE. Our data demon-strated significant disease amelioration from onset to peak evidenced Fig. 5.Brain cytokines. A) Brain TNF levels: EAE-dpi (F2,34= 27.84; Pb0.0001), exercise (F1,34= 42.72; Pb0.0001) and the interaction between both (F2,34= 9.45; P = 0.005) affected

the variance. B) Brain IL-6 levels: EAE-dpi (F2,33= 20.12; Pb0.0001) and exercise (F1,33= 7.04; P = 0.0122) affected the variance. C) Brain IL-1βlevels: EAE-dpi (F2,34= 12.74;

Pb0.0001) and interaction (F2,34= 5.34; P = 0.0096) affected the variance. D) Brain IL-10 levels: EAE-dpi (F2,34= 12.48; Pb0.0001), exercise (F1,34= 11.91; P = 0.0015) and the

interaction between both (F2,34= 3.88; P = 0.0302) affected the variance. Differences between the exercised and unexercised mice were observed in the control group for TNF

(Pb0.001), at 10 dpi-EAE for TNF (Pb0.001), IL-1β(Pb0.01) and IL-10 (Pb0.001) and at 14 dpi-EAE for IL-6 (Pb0.05). The comparisons between the two time points of EAE (10

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by the attenuation of the mean clinical scores and percent weight loss in the exercised EAE versus unexercised EAE groups. Overall, our data are in agreement with previous observations demonstrating the beneficial effect of exercise in EAE (Le Page et al., 1994, 1996; Rossi et al., 2009). In contrast, our investigations were thefirst to focus on the effect of systematic animal swimming exercise protocol in EAE, which appears to be more beneficial than the classic treadmill exercise (Ra et al., 2002; Deforges et al., 2009; Bansi et al., 2012).

EAE is an inflammatory disease of the CNS mediated by CD4+ Th1 cells that serves as animal model of multiple sclerosis (MS). A patholog-ical hallmark of MS is infiltration of immune cells across the blood–brain barrier into the CNS causing myelin destruction and axonal injury (Popescu and Lucchinetti, 2012). To migrate into CNS sites of infl amma-tion, leukocytes mustfirst tether and roll along the venule before they can firmly adhere and emigrate out of the vasculature ( Carvalho-Tavares et al., 2000). To determine whether the exercise-induced clini-cal improvement was associated with leukocyte recruitment to the CNS microcirculation, we assessed in vivo leukocyte rolling and adhesion. These measurements were evaluated in both brain and spinal cord microvasculature in the same animal. However, the data clearly demon-strated that there were no significant differences between the exercised and unexercised EAE groups in the recruitment of leukocytes to the CNS (Figs. 2, 3 and 4).

Previous study reported that no differences in cellular infiltrates were observed at 50 dpi between EAE animals undergoing or not undergoing voluntary exercise (Rossi et al., 2009). Few or any infiltrating T lymphocytes (CD4+ or CD8+ cells) or macrophages (CD68+ cells)

were present in the striatum from both groups of EAE mice (control and exercising) and spinal cord showed similar cellular infiltrates in both EAE groups. The authors then suggested that exercise did not exhibit an anti-inflammatory component in the chronic phase of the disease. The present study is thefirst to address in vivo leukocyte recruitment in the acute phase of the disease, and it demonstrated that the clinical improvement in EAE from exercise was not associated with either a delay or decrease in the inflammatory infiltrate in the CNS.

In order to understand the effects of swimming exercise on molecu-lar immune pomolecu-larization mechanism, we investigated the cytokine profile in the mouse brain and spinal cord, including TNF and IL-1β (proinflammatory), IL-6 (inflammation regulatory) and IL-10 (anti-inflammatory). These cytokines have been implicated in the effects of exercise in MS patients (Dalgas and Stenager, 2012). The anti-inflammatory effect of exercise wasfirst demonstrated in the control group as evidenced by decreased of CNS TNF concentration compared with unexercised controls (Figs. 5A and6A), corroborating previous results, which showed a established effect of regular exercise in healthy animals (Ang et al., 2004). Indeed, it is believed that exercise training has an anti-inflammatory effect because of its sustained IL-6-generating effect (Packer et al., 2010; Gleeson et al., 2011).

In contrast to high brain levels of IL-6, the unexercised EAE mice demonstrated a significant reduction in the other cytokines between 10 and 14 dpi in both brain (Fig. 5) and spinal cord (Fig. 6), which could be associated to the changes observed in the numbers of circulating leukocytes (Fig. 4F). Moreover, the data were consistent with previous data suggesting that the peak expression of cytokines, probably occur Fig. 6.Spinal cord cytokines. A) Spinal cord TNF levels: EAE-dpi (F2,33= 6.26; P = 0.0049), exercise (F1,33= 18.91; Pb0.0001) and the interaction between both (F2,33= 34.18;

Pb0.0001) affected the variance. B) Spinal cord IL-6 levels: EAE-dpi (F2,31= 5.67; P = 0.0080) and the interaction (F2,31= 13.10; Pb0.0001) affected the variance. C) Spinal cord

IL-1βlevels: EAE-dpi (F2,33= 7.00; P = 0.0029) and the interaction (F2,33= 4.88; P = 0.0139) affected the variance. D) Spinal cord IL-10 levels: EAE-dpi (F2,32= 3.70; P = 0.0359),

exercise (F1,32= 5.61; P = 0.0241) and the interaction (F2,32= 6.34; P = 0.0048) affected the variance. Differences between the exercised and unexercised mice were observed at

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by the onset of the disease (for review seeEng et al., 1996). Comparison of brain cytokine levels between EAE-induced exercised and non-exercised groups at 10dpi (Fig. 5), showed significant reduction in TNF, IL-1βand IL-10 levels in the exercised group, thereby demonstrating an overall anti-inflammatory environment. No further change was demon-strated by 14 dpi for these cytokines, but IL-6 levels were significantly reduced over the time period of interest. These data do not support a switch from TH1 to TH2 by the infiltrating cells in the exercised group (Batoulis et al., 2010; El-behi et al., 2010). The unchanged IL-10 level in the exercised group also suggests an absence of the upregulation of reg-ulatory T cells, which are a major source of this cytokine (von Boehmer, 2005). However, IL-6 is required for TH17 cell differentiation (Bettelli et al., 2006), and TH17 cells are important sources of additional IL-6 and TNF (Langrish et al., 2005). Then, the reduction in IL-6 levels in the brains of the exercised EAE mice at 14 dpi may represent a modulatory mecha-nism of the TH17 response in the CNS. Additional study will be necessary in order to test this hypothesis.

Surprisingly, different profiles were observed between CNS regions. In the spinal cord of the exercised EAE mice, an upregulation of TNF, IL-6 and IL-10 at 14 dpi was observed relative to the unexercised group (Fig. 6A, B and D, respectively), demonstrating the generation of a pro-inflammatory environment in this CNS region. In addition, this response occurred in the absence of changes in the circulating leukocyte number (Fig. 4F) or the spinal cord recruitment of leukocytes (Figs. 2C, D,3D and4E). Interestingly, as shown inFig. 6, the data suggest that this potential exercise effect is a specific response triggered after EAE induction because the exercised control group did not exhibit a similar

response. Indeed, it has been demonstrated that 6 weeks of regular, moderate-intensity exercise prior to vaccination for hepatitis B antigen enhances immune functions by increasing pro-inflammatory cytokines and antigen-specific cell-mediated immunity in mice (Wang et al., 2011). Then, although no differences were observed between the exercised and unexercised mice for recruitment of leukocytes, the cyto-kine data suggest that swimming exercise is associated with enhanced immune function at the CNS site (spinal cord) more severely damaged in the EAE animals.

TNF, IL-1βand IL-6 are typically linked to tissue destruction, and they have been classically associated with EAE disability (Batoulis et al., 2010). The combination of these cytokines with decreased BDNF trophic support has been shown to result in neuronal apoptosis in a spinal cord injury model (Basso and Hansen, 2011). However, there is a strong body of evidence supporting the concept that CNS infl amma-tion has both destructive and beneficial effects (Hohlfeld et al., 2007). Protective effects are related to the BDNF produced and released by neu-rons and leukocytes (Kerschensteiner et al., 1999; Zhu et al., 2012). EAE mice has demonstrated elevated BDNF in the brain at peak disease which could reflect self-repair mechanisms followed by a decline that is indicative of the disease damage (Aharoni et al., 2005). However, when these EAE mice were treated with glatiramer acetate, BNDF levels do not decline and it was shown a co localization of the neurotrophic factor with lymphocytes (CD3), astrocytes (GFAP) and neurons (NeuN). It is important to note that BDNF stimulation has been suggested to have therapeutic effects in EAE (Makar et al., 2009) as well as in MS (Azoulay et al., 2008; Comini-Frota et al., 2012).

In our study, we observed increased BDNF levels in both the brain and spinal cord homogenates from exercised EAE mice compared with Fig. 7.Brain and spinal cord BDNF. A) Brain BDNF levels: EAE-dpi alone (F2,32= 5.15;

P = 0.0115) and the interaction between exercise and EAE (F2,32= 13.14; Pb0.0001)

affected the variance. B) Spinal cord BDNF levels: EAE-dpi (F2,33= 23.26; Pb0.0001),

exercise (F1,33= 8.61; P = 0.0061) and the interaction between both factors (F2,33=

10.65; P = 0.0003) affected the variance. Differences between the exercised and unexer-cised mice were observed in the control group for the spinal cord (Pb0.05), at 10 day pi-EAE for the brain (Pb0.05) and at 14 dpi-EAE for the brain (Pb0.001) and spinal cord (Pb0.001). The comparisons between the two time points of the EAE (10 and

14 dpi) and control mice subjected to the same exercise condition (exercised or unexer-cised) were performed using one-way ANOVA with the Tukey post-test and are represented by the capped lines.

Fig. 8.Demyelination pathological analysis of exercised and unexercised EAE mice. Images of LFB-stained spinal cord (A, B) at 14 dpi (representative of 5 mice per group). Note the demyelination lesion in the anterior funiculum of spinal cord in both the unexercised (A) and exercised (B) EAE mice. C) Optical density quantification of a single LFB-stained spinal cord section from each mouse. Differences were observed between control and 14 dpi and between 10 and between 14 dpi only for the unexercised mice (Pb0.01).

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unexercised EAE mice at 14 dpi. This result could represent an improve-ment in the repair process and affect myelin degeneration by prolifera-tion and/or regeneraprolifera-tion of oligodendrocytes within lesions area (Skihar et al., 2009).

Recent study indicated that cerebrospinalfluid derived from primary progressive MS patients reduced the proliferation of human embryonic-derived neural precursor cells and increased their differentiation toward neuronal and oligodendroglial cells (Cristofanilli et al., 2013). In this sense, as it has been observed higher serum levels of BDNF during MS relapse (Sarichielli et al., 2002), strategies to improve BDNF release during relapse could promote the addictive neuroprotective effect.

Thus, in order to support the attenuation of the mean clinical scores as well as increase of BDNF levels in the exercised EAE mice, the levels of demyelination have been assessed. And, in fact, the progression of spinal cord demyelination from 10 to 14 dpi was less pronounced in the exercised EAE mice (Fig. 8C). Cumulatively, although no significant, the lesion size and the total volume of demyelination were about 40% and 30% smaller, respectively, in the exercised group compared to the unexercised group. These results supported the 36% attenuation in the clinical score (Pb0.001; area under curve of each group compared by

Mann Whitney test).

It is important to note that white matter pathology in MOG-EAE has been correlated with clinical disease severity and a transition from mainly inflammatory processes to demyelination and axonal damage from acute to chronic time point has been assumed (Recks et al., 2011). A 7-day treatment with glatiramer acetate has been shown to increase BDNF production by leukocytes and this result was associated with remyelination after 20 days of the treatment cessation (Skihar et al., 2009). Therefore, additional investigation of the effects of prior swimming exercise in the chronic phase of EAE could provide some interesting information.

In summary, the present study reveals for thefirst time that prior swimming exercise attenuated clinical presentation of EAE from onset to peak disease and reduced demyelination, probably via increase of BDNF. Because BDNF is involved in the myelinogenesis it will be relevant to determine the mechanism underlying BDNF-induced myelination after exercise. In conclusion, our data strongly support the hypothesis that release of neurotrophins such as BDNF might be implicated in the clinical as well as in the neuropathological effects of exercise in EAE.

Acknowledgments

We are grateful to Pedro William Machado de Almeida and Silvia Carolina Guatimosim Fonseca for their permission to use the swimming pool, to Daisy Motta for the advice on the protocol for workload testing and to Antonio Lucio Teixeira for generously providing the BDNF antibod-ies. This work is part of DB's PhD thesis and receivedfinancial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), Fundação do Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG, Brazil), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (PDSE-CAPES, Brazil, BEX 0020/12-5). JRB and RB were supported by NIH Funding (NS051709, NS065479) and by The Miami Project To Cure Paralysis. JCT was supported by FAPEMIG (grant CBB-APQ-01459-10) and Pró-Reitoria de Pesquisa da UFMG (PRPq/ UFMG). RMEA receives a research fellowship from CNPq, andfinancial support from FAPEMIG (PPM-00200-12) and CNPq (473013/2012-6).

References

Aharoni, R., Eilam, R., Domev, H., Labunskay, G., Sela, M., Arnon, R., 2005.The immunomodulator glatiramer acetate augments the expression of neurotrophic factors in brains of experimental autoimmune encephalomyelitis mice. PNAS 102, 19045–19050.

Almeida, P.W., Gomes-Filho, A., Ferreira, A.J., Rodrigues, C.E., Dias-Peixoto, M.F., Russo, R.C., Teixeira, M.M., Cassali, G.D., Ferreira, E., Santos, I.C., Garcia, A.M., Silami-Garcia,

E., Wisloff, U., Pussieldi, G.A., 2009.Swim training suppresses tumor growth in mice. J. Appl. Physiol. 107, 261–265.

Ang, E.T., Gomez-Pinilla, F., 2007.Potential therapeutic effects of exercise to the brain. Curr. Med. Chem. 14, 2564–2571.

Ang, E.T., Wong, P.T., Moochhala, S., Ng, Y.K., 2004.Cytokine changes in the horizontal diagonal band of Broca in the septum after running and stroke: a correlation to glial activation. Neuroscience 129, 337–347.

Azoulay, D., Urshansky, N., Karni, A., 2008.Low and dysregulated BDNF secretion from immune cells of MS patients is related to reduced neuroprotection. J. Neuroimmunol. 195, 186–193.

Bansi, J., Bloch, W., Gamper, U., Kesselring, J., 2012.Training in MS: influence of two different endurance training protocols (aquatic versus overland) on cytokine and neurotrophin concentrations during three week randomized controlled trial. Mult. Scler. 1–9.

Basso, M., Hansen, C.N., 2011.Biological basis of exercise-based treatments: spinal cord injury. Phys. Med. Rehabil. 3, S73–S77.

Batoulis, H., Addicks, K., Kuerten, S., 2010.Emerging concepts in autoimmune encephalo-myelitis beyond the CD4/T(H)1 paradigm. Ann. Anat. 192, 179–193.

Bettelli, E., Carrier, Y., Gao, W., Korn, T., Strom, T.B., Oukka, M., Weiner, H.L., Kuchroo, V.K., 2006.Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441, 235–238.

Carvalho-Tavares, J., Hickey, M.J., Hutchison, J., Michaud, J., Sutcliffe, I.T., Kubes, P., 2000.A role for platelets and endothelial selectins in tumor necrosis factor-{alpha}-induced leukocyte recruitment in the brain microvasculature. Circ. Res. 87, 1141–1148 (Am Heart Assoc).

Castellano, V., White, L.J., 2008.Serum brain-derived neurotrophic factor response to aerobic exercise in multiple sclerosis. J. Neurol. Sci. 269, 85–91.

Castellano, V., Patel, D.I., White, L.J., 2008.Cytokine responses to acute and chronic exercise in multiple sclerosis. J. Appl. Physiol. 104, 1697–1702.

Comini-Frota, E.R., Rodrigues, D.H., Miranda, E.C., Brum, D.G., Kaimen-Maciel, D.R., Donadi, E.A., Teixeira, A.L., 2012.Serum levels of brain-derived neurotrophic factor correlate with the number of T2 MRI lesions in multiple sclerosis. Braz. J. Med. Biol. Res. 45, 68–71.

Cristofanilli, M., Cymring, B., Lu, A., Rosenthal, H., Sadiq, S.A., 2013. Cerebrospinalfluid derived from progressive multiple sclerosis patients promotes neuronal and oligo-dendroglial differentiation of human neural precursor cells in vitro. Neuroscience S0306-4522 (13). http://dx.doi.org/10.1016/j.neuroscience.2013.07.022 (00604-0, [pii]).

Dalgas, U., Stenager, E., 2012.Exercise and disease progression in multiple sclerosis: can exercise slow down the progression of multiple sclerosis? Ther. Adv. Neurol. Disord. 5, 81–95.

De Santi, L., Annunziata, P., Sessa, E., Bramanti, P., 2009.Brain-derived neurotrophic factor and TrkB receptor in experimental autoimmune encephalomyelitis and multiple sclerosis. J. Neurol. Sci. 287, 17–26.

De Santi, L., Polimeni, G., Cuzzocrea, S., Esposito, E., Sessa, E., Annunziata, P., Bramanti, P., 2011.Neuroinflammation and neuroprotection: an update on (future) neurotrophin-related strategies in multiple sclerosis treatment. Curr. Med. Chem. 18, 1775–1784.

Deforges, S., Branchu, J., Biondi, O., Grondard, C., Pariset, C., Lecolle, S., Lopes, P., Vidal, P.P., Chanoine, C., Charbonnier, F., 2009.Motoneuron survival is promoted by specific exercise in a mouse model of amyotrophic lateral sclerosis. J. Physiol. 587, 3561–3572.

dos Santos, A.C., Barsante, M.M., Arantes, R.M., Bernard, C.C., Teixeira, M.M., Carvalho-Tavares, J., 2005.CCL2 and CCL5 mediate leukocyte adhesion in experimental autoim-mune encephalomyelitis—an intravital microscopy study. J. Neuroimmunol. 162, 122–129.

dos Santos, A.C., Roffe, E., Arantes, R.M., Juliano, L., Pesquero, J.L., Pesquero, J.B., Bader, M., Teixeira, M.M., Carvalho-Tavares, J., 2008.Kinin B2 receptor regulates chemokines CCL2 and CCL5 expression and modulates leukocyte recruitment and pathology in experimental autoimmune encephalomyelitis (EAE) in mice. J. Neuroinflammation 5, 49.

El-behi, M., Rostami, A., Ciric, B., 2010.Current views on the roles of Th1 and Th17 cells in experimental autoimmune encephalomyelitis. J. Neuroimmune Pharmacol. 5, 189–197.

Eng, L.F., Ghirnikar, R.S., Lee, Y.L., 1996.Inflammation in EAE: role of chemokine/cytokine expression by resident and infiltrating cells. Neurochem. Res. 21, 511–525.

Evangelista, F.S., Brum, P.C., Krieger, J.E., 2003.Duration-controlled swimming exercise training induces cardiac hypertrophy in mice. Braz. J. Med. Biol. Res. 36, 1751–1759.

Gleeson, M., Bishop, N.C., Stensel, D.J., Lindley, M.R., Mastana, S.S., Nimmo, M.A., 2011.

The anti-inflammatory effects of exercise: mechanisms and implications for the prevention and treatment of disease. Nat. Rev. Immunol. 11, 607–615.

Gold, S.M., Schulz, K.H., Hartmann, S., Mladek, M., Lang, U.E., Hellweg, R., Reer, R., Braumann, K.M., Heesen, C., 2003.Basal serum levels and reactivity of nerve growth factor and brain-derived neurotrophic factor to standardized acute exercise in multiple sclerosis and controls. J. Neuroimmunol. 138, 99–105.

Golzari, Z., Shabkhiz, F., Soudi, S., Kordi, M.R., Hashemi, S.M., 2010.Combined exercise training reduces IFN-gamma and IL-17 levels in the plasma and the supernatant of peripheral blood mononuclear cells in women with multiple sclerosis. Int. Immunopharmacol. 10, 1415–1419.

Heesen, C., Gold, S.M., Hartmann, S., Mladek, M., Reer, R., Braumann, K.M., Wiedemann, K., Schulz, K.H., 2003.Endocrine and cytokine responses to standardized physical stress in multiple sclerosis. Brain Behav. Immun. 17, 473–481.

Hohlfeld, R., Kerschensteiner, M., Meinl, E., 2007.Dual role of inflammation in CNS disease. Neurology 68, S58–S63 (discussion S91-56).

Kerfoot, S.M., Kubes, P., 2002.Overlapping roles of P-selectin and alpha 4 integrin to recruit leukocytes to the central nervous system in experimental autoimmune encephalomyelitis. J. Immunol. 169, 1000–1006.

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H., Wekerle, H., Hohlfeld, R., 1999.Activated human T cells, B cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? J. Exp. Med. 189, 865–870.

Langrish, C.L., Chen, Y., Blumenschein, W.M., Mattson, J., Basham, B., Sedgwick, J.D., McClanahan, T., Kastelein, R.A., Cua, D.J., 2005.IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. Exp. Med. 201, 233–240.

Le Page, C., Ferry, A., Rieu, M., 1994.Effect of muscular exercise on chronic relapsing experimental autoimmune encephalomyelitis. J. Appl. Physiol. 77, 2341–2347.

Le Page, C., Bourdoulous, S., Beraud, E., Couraud, P.O., Rieu, M., Ferry, A., 1996.Effect of physical exercise on adoptive experimental auto-immune encephalomyelitis in rats. Eur. J. Appl. Physiol. Occup. Physiol. 73, 130–135.

Luhder, F., Gold, R., Flugel, A., Linker, R.A., 2013.Brain-derived neurotrophic factor in neuroimmunology: lessons learned from multiple sclerosis patients and experimental autoimmune encephalomyelitis models. Arch. Immunol. Ther. Exp. (Warsz.) 61, 95–105.

Makar, T.K., Bever, C.T., Singh, I.S., Royal, W., Sahu, S.N., Sura, T.P., Sultana, S., Sura, K.T., Patel, N., Dhib-Jalbut, S., Trisler, D., 2009.Brain-derived neurotrophic factor gene delivery in an animal model of multiple sclerosis using bone marrow stem cells as a vehicle. J. Neuroimmunol. 210, 40–51.

Motl, R.W., Pilutti, L.A., 2012.The benefits of exercise training in multiple sclerosis. Nat. Rev. Neurol. 8, 487–497.

Odoardi, F., Kawakami, N., Klinkert, W.E., Wekerle, H., Flugel, A., 2007.Blood-borne soluble protein antigen intensifies T cell activation in autoimmune CNS lesions and exacerbates clinical disease. Proc. Natl. Acad. Sci. U. S. A. 104, 18625–18630.

Oh-ishi, S., Kizaki, T., Toshinai, K., Haga, S., Fukuda, K., Nagata, N., Ohno, H., 1996.

Swimming training improves brown-adipose-tissue activity in young and old mice. Mech. Ageing Dev. 89, 67–78.

Packer, N., Pervaiz, N., Hoffman-Goetz, L., 2010.Does exercise protect from cognitive decline by altering brain cytokine and apoptotic protein levels? A systematic review of the literature. Exerc. Immunol. Rev. 16, 138–162.

Patel, D.I., White, L.J., 2013.Effect of 10-day forced treadmill training on neurotrophic factors in experimental autoimmune encephalomyelitis. Appl. Physiol. Nutr. Metab. 38, 194–199.

Petratos, S., Ozturk, E., Azari, M.F., Kenny, R., Lee, J.Y., Magee, K.A., Harvey, A.R., McDonald, C., Taghian, K., Moussa, L., Mun Aui, P., Siatskas, C., Litwak, S., Fehlings, M.G., Strittmatter, S.M., Bernard, C.C., 2012.Limiting multiple sclerosis related axonopathy by blocking Nogo receptor and CRMP-2 phosphorylation. Brain 135, 1794–1818.

Popescu, B.F., Lucchinetti, C.F., 2012.Pathology of demyelinating diseases. Annu. Rev. Pathol. 7, 185–217.

Ra, S.M., Kim, H., Jang, M.H., Shin, M.C., Lee, T.H., Lim, B.V., Kim, C.J., Kim, E.H., Kim, K.M., Kim, S.S., 2002.Treadmill running and swimming increase cell proliferation in the hippocampal dentate gyrus of rats. Neurosci. Lett. 333, 123–126.

Recks, M.S., Addicks, K., Kuerten, S., 2011.Spinal cord histopathology of MOG peptide 35–55-induced experimental autoimmune encephalomyelitis is time- and score-dependent. Neurosci. Lett. 494, 227–231.

Rossi, S., Furlan, R., De Chiara, V., Musella, A., Lo Giudice, T., Mataluni, G., Cavasinni, F., Cantarella, C., Bernardi, G., Muzio, L., Martorana, A., Martino, G., Centonze, D., 2009.

Exercise attenuates the clinical, synaptic and dendritic abnormalities of experimental autoimmune encephalomyelitis. Neurobiol. Dis. 36, 51–59.

Sarichielli, P., Greco, L., Stipa, A., Floridi, A., Gallai, V., 2002.Brain-derived neurotrophic factor in patients with multiple sclerosis. J. Neuroimmunol. 132, 180–188.

Schulz, K.H., Gold, S.M., Witte, J., Bartsch, K., Lang, U.E., Hellweg, R., Reer, R., Braumann, K.M., Heesen, C., 2004.Impact of aerobic training on immune-endocrine parameters, neurotrophic factors, quality of life and coordinative function in multiple sclerosis. J. Neurol. Sci. 225, 11–18.

Sigwalt, A.R., Budde, H., Helmich, I., Glaser, V., Ghisoni, K., Lanza, S., Cadore, E.L., Lhullier, F.L.R., Bem, A.F.d, Hohl, A., Matos, F.J.d, Oliveira, P.A.d, Prediger, R.D., Guglielmo, L.G.A., Latini, A.A., 2011. Molecular aspects involved in swimming exercise training reducing anhedonia in a rat model of depression. Neuroscience 192, 661–674.

Skihar, V., Silva, C., Chojnacki, A., Doring, A., Stallcup, W.B., Weiss, S., Yong, V.W., 2009.

Promoting oligodendrogenesis and myelin repair using the multiple sclerosis medication glatiramer acetate. Proc. Natl. Acad. Sci. U. S. A. 106, 17992–17997.

Sung, C., Chiu, C.-Y., Lee, E.-J., Bezyak, J., Chan, F., Muller, V., 2013.Exercise, diet, and stress management as mediators between functional disability and health-related quality of life in multiple sclerosis. Rehabil. Couns. Bull. 56 (2), 85–95.

von Boehmer, H., 2005.Mechanisms of suppression by suppressor T cells. Nat. Immunol. 6, 338–344.

Wang, J., Song, H., Tang, X., Yang, Y., Vieira, V.J., Niu, Y., Ma, Y., 2011.Effect of exercise training intensity on murine T-regulatory cells and vaccination response. Scand. J. Med. Sci. Sports 22, 643–652.

Waschbisch, A., Wenny, I., Tallner, A., Schwab, S., Pfeifer, K., Mäurer, M., 2012.

Physical activity in multiple sclerosis: a comparative study of vitamin D, brain-derived neurotrophic factorand regulatory T cell populations. Eur. Neurol. 68, 122–128.

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