Moderate physical exercise reduces parasitaemia and protects colonic myenteric neurons in mice infected with Trypanosoma cruzi

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1 Int. J. Exp. Path. (2013), 94, ORIGINAL ARTICLE Moderate physical exercise reduces parasitaemia and protects colonic myenteric neurons in mice infected with Trypanosoma cruzi Neide M. Moreira*, Franciele d. N. Santos, Max Jean d. O. Toledo, Solange M. F. d. Moraes, Eduardo J. d. A. Araujo, Debora d. M. G. Sant Ana** and Silvana M. d. Araujo *Programa de Pos-Graduacß~ao em Ci^encias da Saude, Universidade Estadual de Maringa, Maringa, Parana, Brazil, Mestranda pela Escola de Enfermagem da Universidade de S~ao Paulo, Avenida. Dr. Eneas Carvalho de Aguiar, S~ao Paulo, Brazil, Departamento de Ci^encias Basicas da Saude Parasitologia, Universidade Estadual de Maringa, Maringa, Parana, Brazil, Departamento de Ci^encias fisiologicas, Universidade Estadual de Maringa, Maringa, Parana, Brazil, Departamento de Histologia, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Campus Universitario, Parana, Brazil and **Departamento de Ci^encias Morfologicas Programa de Pos-Graduacß~ao em Bioci^encias Aplicadas a Farmacia, Universidade Estadual de Maringa, Maringa, Parana, Brazil INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY doi: /iep Received for publication: 23 April 2013 Accepted for publication: 28 July 2013 Correspondence Neide M. Moreira Departamento de Ci^encias Basicas da Saude, Setor de Parasitologia Universidade Estadual de Maringa Avenida Colombo, 5790 Zona 07, , Maringa Parana Brazil Tel.: (55 44) Fax: (55 44) neidemartinsenf@yahoo. com.br SUMMARY This study evaluated the influence of moderate physical exercise on the myenteric neurons in the colonic intestinal wall of mice that had been infected with Trypanosoma cruzi. Parasitology and immunological aspects of the mice were considered. Forty-day-old male Swiss mice were divided into four groups: Trained Infected (TI), Sedentary Infected (SI), Trained Control (TC), and Sedentary Control (SC). The TC and TI were subjected to a moderate physical exercise program on a treadmill for 8 weeks. Three days after finishing exercise, the TI and SI groups were inoculated with 1,300 blood trypomastigotes of the Y strain-t. cruzi. After 75 days of infection results were obtained. Kruskal-Wallis or Analyze of variance (Tukey post hoc test) at 5% level of significance was performed. Moderate physical exercise reduced both the parasite peak (day 8 of infection) and total parasitemia compared with the sedentary groups (P < 0.05). This activity also contributed to neuronal survival (P < 0.05). Exercise caused neuronal hypertrophy (P < 0.05) and an increase in the total thickness of the intestinal wall (P < 0.05). The TI group exhibited an increase in the number of intraepithelial lymphocytes (P > 0.05). In trained animals, the number of goblet cells was reduced compared with sedentary animals (P < 0.05). Physical exercise prevented the formation of inflammatory foci in the TI group (P < 0.05) and increased the synthesis of TNF-a (P < 0.05) and TGF-b (P > 0.05). The present results demonstrated the benefits of moderate physical exercise, and reaffirmed the possibility of that it may contribute to improving clinical treatment in Chagas disease patients. Keywords colon, myenteric plexus, moderate physical exercise, physical training, TNF-a, TGF-b, Trypanosoma cruzi Chagas disease is caused by Trypanosoma cruzi. It is a serious public health problem in countries with a high prevalence (Schmunis & Yadon 2010). Approximately 10 million people worldwide are infected with this parasite, especially in Latin America where the disease is endemic, and more than 25 million people are at risk of infection (World Health Organization 2013). Nearly 3 million people are currently infected in Brazil (Portal of Health 2013). During the course of the disease, the parasite invades a wide variety of organs, including the heart, central nervous system, intes The Authors. 426 International Journal of Experimental Pathology 2013 International Journal of Experimental Pathology

2 Physical training 9 neurons 9 T. cruzi 427 tines and oesophagus (Lana & Tafure 2011). Megaesophagus and megacolon are described as primary manifestations of the disease that result from gastrointestinal tract lesions (Pallisera et al. 2011). Motility disorders are also associated with the disease, with constipation and dilatation of the viscera with or without thickening of the intestinal wall (Silveira et al. 2007). These processes arise from the death of neurons in the enteric nervous system (Silveira et al. 2007; Lana & Tafure 2011; Pallisera et al. 2011). The aetiological treatment of Chagas disease is based on two drugs, benznidazole and nifurtimox, which have limited efficacy in the chronic phase (i.e. the condition of most infected individuals; Fabbro et al. 2007; Hasslocher-Moreno et al. 2012). Therefore, patients who undergo aetiological treatment may have symptoms of severe constipation and intestinal complications (Santos Junior 2002). The search for alternative interventions that seek to improve the management of these patients is a major challenge. The Chagas Disease Laboratory at the State University of Maringa seeks to improve the care of patients infected by T. cruzi (Araujo et al. 2000). The laboratory has invested in basic and applied research with this purpose (Schebeleski-Soares et al. 2009). Previous studies showed that chronic, moderate physical exercise (i.e. treadmill exercise) contributes to maintaining the number of myenteric neurons (De Britto Mari et al. 2008; Martinez Gagliardo et al. 2008). Such exercise has also been shown to be a resistance factor in the development of infections caused by protozoa in animals (Soares et al. 2010; Occhi et al. 2012). Chronic, moderate physical exercise stimulates the immune response (Nakamura et al. 2010; Soares et al. 2010) and serves as a positive modulator when considering neuroimmunoendocrine changes in patients with chronic heart failure (Rosa & Junior 2005). Research conducted in animals has revealed positive effects of chronic, moderate physical exercise (i.e. treadmill exercise) on experimental T. cruzi infection (Schebeleski-Soares et al. 2009; Soares et al. 2010; Occhi et al. 2012). However, no studies have evaluated the changes produced in the myenteric plexus and colon wall in mice infected with T. cruzi and subjected to physical exercise. The present study investigated the influence of chronic, moderate physical exercise (i.e. treadmill exercise) on myenteric neurons and the wall of the colon in mice infected with T. cruzi by examining parasitological and immunological aspects. Materials and methods Animals The experiment was performed as a controlled, randomized, blind trial and repeated twice. In each replication, forty 30- day-old male Swiss mice were used and divided into two groups: trained (n = 20) and sedentary (n = 20). The animals were conditioned in polypropylene boxes ( mm) closed with a galvanized grid with central depression for offering food and water. The boxes were lined with wood shavings and cleaned twice weekly. The boxes remained in the conditioning room (temperature, C) under a 12 h/12 h light/dark cycle. The animals were given ad libitum access to chlorinated drinking water and food (Nuvilab Cr-1, Nuvital). Ethical approval The present study was endorsed by the Ethics Committee on Animal Experiments (ECAE), State University of Maringa, Brazil (no. 046/2009). Moderate physical exercise protocol The 30-day-old animals were subjected to an aerobic physical exercise programme on a treadmill (Inbrasport Classic CI model; Petropolis, Rio de janeiro, Brazil) for 8 weeks. The exercise consisted of a daily training session five times per week that included the following: min per session at a speed of 6 14 m/min in the first week, min per session at a speed of 8 16 m/min in the second week and 60 min at a speed of m/min in the subsequent weeks. The mean speed was 13 m/min in the first 4 weeks and 17.5 m/min in the last 4 weeks. The sessions began at 6:00 pm during the dark/active phase of the light/dark cycle. The temperature was maintained at C during the training sessions. The physical exercise protocol used is considered to require light or mild effort (Lerman et al. 2002; Schebeleski-Soares et al. 2009). The treadmill had an adapter for small-animal training and a system that allowed both training-session planning and digital speed control with 2 m/min sensitivity. The training was conducted in the Exertion Physiology Laboratory, Department of Physiological Sciences, State University of Maringa. Shock or similar mechanisms were not used to induce the animals to exercise, and a cardboard support was adapted to the top of each lane so that the animals could rest during training when they felt tired. Infection After completing the physical exercise programme, the animals were assigned to four groups: trained control (TC; subjected to physical exercise and uninfected; n = 10), trained infected (TI; subjected to physical exercise and subsequently infected; n = 10), sedentary control (SC; not subjected to physical exercise and uninfected; n = 10) and sedentary infected (SI; not subjected to physical exercise and subsequently infected; n = 10). An intraperitoneal inoculum of 1300 bloodstream forms of the Y strain of T. cruzi was used (Moreira et al. 2011). The TI and SI groups were inoculated 3 days after the physical exercise programme. Chronic infection was obtained with five doses of benznidazole (Rochagan; LAFEPE, Pernambuco, Brazil; three doses of 100 mg/kg, m.c., 11, 15 and 22 days after inoculation; two doses of 250 mg/kg,

3 428 N. M. Moreira et al. m.c., 18 and 41 days after injection) via oral gavage (adapted from Moreira et al. 2011). Evaluation of the course of infection Parasitaemia was evaluated in all of the infected animals using Brener s technique (1962). Parasite scores were recorded daily from day 4 to day 15 after inoculation and on alternate days from day 15 to day 61 after inoculation. The curve was traced using the mean parasitaemia of the inoculated animals for each group. The following averages were obtained: prepatent period (mean time in days from the day of experimental inoculation to the day in which positivity was observed with the fresh blood examination for each group), patent period (mean time in days that each group had parasitaemia observed with the fresh blood examination), parasite peak (the largest number of parasites observed in each group from the curve of mean parasitaemia), total parasitaemia (the sum average of the parasite number of each animal), mortality (this was recorded throughout the experiment for a period of 75 days) and animal body mass (this was checked once per week throughout the experiment). Euthanasia and organ collection Seventy-five days after inoculation, all of the animals were euthanized with an overdose saturated camera with diethyl ether (Moreira et al. 2011). The colon of five animals from each group was removed, washed with 0.85% saline solution and filled and immersed in formaldehyde acid fixative solution. After 48 h, the colons were dissected to prepare the muscularis and serosa tunica and stained using Giemsa s technique (Barbosa 1978) to allow the observation of the total myenteric neuron population, which was counted and measured. The colon area (expressed in cm 2 ) was calculated based on the width and length of each organ collected. Quantitative analysis of myenteric neurons The neurons present in 40 microscopic fields in each region (i.e. mesenteric, intermediate and antimesenteric) were quantified from a total of 120 fields across the intestinal circumference in each animal. A photonic microscope (Olympus CBA, S~ao Paulo, Brazil) was used, with a 409 objective. Denervation was assessed by comparing the experimental and control groups and is expressed as a percentage. Morphometric analysis of myenteric neurons The area (lm 2 ) of both the cell bodies and nuclei of 100 neurons of the myenteric plexus in each region (i.e. mesenteric, intermediate and antimesenteric) was measured from a total of 300 neurons distributed throughout the intestinal circumference in each animal. The images were taken by a trinocular photonic microscope (Motic B5; Ribeir~ao Preto, S~ao Paulo, Brazil) coupled to a high-definition digital video camera (Moticam 2000; Ribeir~ao Preto, S~ao Paulo, Brazil). Measurements were made using IMAGEPRO PLUS software. Histological analysis of the intestinal wall Three 4-lm-thick histological cross-sections of the intestinal wall were obtained from five animals in each group and stained with haematoxylin eosin (HE) to quantify intraepithelial lymphocytes (IELs) and inflammatory foci. To analyse goblet cells, periodic acid-schiff (PAS) was used to detect neutral mucins. Alcian blue (AB) ph 2.5+ was used to detect sialomucins/sulphomucins, and AB ph 1.0+ was used to detect sulphomucins (Myers et al. 2008). Counterstaining with haematoxylin was performed for PAS and AB staining. Morphometric analysis of the intestinal wall Images were captured under 2009 magnification to measure the tunica mucosa thickness, the depth and width of the crypt, the submucosal tunica and the muscularis. Images were captured under 409 magnification to measure the total wall thickness. Images were captured under magnification to measure the enterocyte height and their nuclei area. Eighty measurements were made for each structure, which were evenly distributed around the intestinal circumference of each mouse (Braga et al. 2011). The sections were stained with HE, and the images were captured using a digital camera (Moticam 2000, 2.0 Megapixel) coupled to a trinocular light microscope (MOTIC B5). Quantitative analysis of intraepithelial lymphocytes, goblet cells, and inflammatory foci Two thousand five hundred epithelial cells from each animal were quantified. The ratios of lymphocytes/100 epithelial cells (Chott et al. 1997) and goblet cells/100 epithelial cells (Hernandes et al. 2003) were calculated using sections stained with HE, PAS, AB ph 2.5+ and AB ph We further quantified inflammatory foci and the number of inflammatory cells within the focus field in 10 microscopic fields with an area of 1.9 mm² distributed across the intestinal circumference in the tunica muscularis of three histological sections of each animal to obtain the average. Groups with 10 or more inflammatory cells were considered inflammatory foci (Oliveira et al. 2007). A photonic microscope (Olympus CBA, S~ao Paulo, Brazil) was used, with 4009 magnification. Cytokine dose The cytokines interleukin-10 (IL-10), interferon-c (IFN-c), tumour necrosis factor-a (TNF-a) and transforming growth factor-b (TGF-b) were dosed in the plasma in 10 animals per group using the enzyme-linked immunosorbent assay (ELISA) capture technique, with antibody pairs from R&D

4 Physical training 9 neurons 9 T. cruzi 429 Systems (Minneapolis, MN, USA). The concentrations of the cytokines were determined by considering the standard curve obtained with recombinant murine cytokines. The results are expressed in pg/ml for IL-10, IFN-c and TNF-a and in ng/ml for TGF-b. Statistical analysis The distribution of the data was verified by applying the D Agostino Pearson or Shapiro Wilk test. Data with a normal distribution are expressed as mean SD and were compared using analysis of variance (ANOVA), followed by the Tukey post hoc test. Unspecified distribution data are expressed as medians and 25th and 75th percentiles and were compared using the Kruskal Wallis test and median test. A 5% significance level and BIOESTAT 5.0 software were used. The statistical effect ( accessed June 26, 2013) was determined using the effect size calculator test by checking the intensity of the effect of physical exercise compared with sedentarism in mice infected with T. cruzi. The effect was considered small when an effect 0.2 was observed. The effect was considered medium when an effect 0.5 was observed. The effect was considered large when an effect >0.5 was observed. Results Course of the infection The parasitaemia curve showed a characteristic profile of the Y strain of T. cruzi in both infected groups. The parasite peak on day 8 of infection and total parasitaemia were 60.0% (P < 0.05) and 44.3% (P < 0.05) lower in the TI group than in the SI group. No significant difference in infectivity, the prepatent period, the patent period or mortality was found between groups (P > 0.05; Figure 1, Table 1). During infection, the animal body mass remained stable in both groups (P > 0.05). No significant changes in the length, diameter or total area of the colon were observed (P > 0.05). Quantitative analysis of the total myenteric neuronal population Table 2 and Figure 2 show the comparison of the total myenteric neuronal population between groups. Moderate physical exercise protected the total myenteric neuronal population by 17.1% These data were obtained by subtracting the comparison percentage of trained animals (TC 9 TI = 22.0%; P < 0.05) from the comparison percentage of sedentary animals (SI 9 SC = 39.1%; P < 0.05). The analysis of the number of neurons in the total colon area indicated that moderate physical exercise significantly prevented neuronal death in the total myenteric neuronal population by 8.0%. These data were obtained by subtracting the comparison percentage of trained animals (TI 9 TC = 36.3%; P < 0.05) from the comparison percentage of sedentary animals (SI 9 SC = 44.1%; P < 0.05; Table 2, Figure 2). Neuronal protection was confirmed by the statistical effect that showed a large effect of 1.1 for the comparison (TI 9 SI). Figure 1 Mean parasitaemia curve of 12-week-old male Swiss mice subjected to moderate physical exercise and subsequently infected (TI group) or not subjected to physical exercise (sedentary) and subsequently infected (SI group). Infection occurred with 1300 blood trypomastigotes of the Y strain of T. cruzi. *, **P < 0.05, comparison between TI and SI group on day 8 after infection (Mann Whitney test). Morphometric analysis of the total myenteric neuronal population Moderate physical exercise and infection in trained animals (TI 9 TC) increased the nuclear area by 30.0%, the cytoplasmic area by 82.1% and the neuronal body area by 59.2% (P < 0.05). In sedentary animals (SI 9 SC), the infection Table 1 Parasitological parameters evaluated in 12-week-old male Swiss mice subjected to physical exercise and subsequently infected (TI) or not subjected to exercise and subsequently infected (SI) with 1300 blood trypomastigotes of the Y strain of T. cruzi for 75 days Group Infectivity (%) Prepatent period (days) Patent period (days) Parasite peak (trypomastigotes/ml ) Total parasitaemia (trypomastigotes/ml ) TI (n = 10) a a 4.6 SI (n = 10) b b 9.5 n, number of animals. The data are expressed as mean standard deviation. Different letters in the same column indicate significant difference (P < 0.05; Mann Whitney test).

5 430 N. M. Moreira et al. Table 2 Neuronal myenteric density of the colon in 240 microscopic fields with an area of 45.6 mm 2 in 22-week-old male Swiss mice. Infection occurred with 1300 blood trypomastigotes of the Y strain of T. cruzi for 75 days Group TC (n = 5) TI (n = 5) SC (n = 5) SI (n = 5) Neuronal density in 45.6 mm 2 10,049.3 a b b c Projection of the number of neurons for the colon area d e f g 21.0 n, number of animals; TC, trained control; TI, trained infected; SC, sedentary control; SI, sedentary infected. The data are expressed as mean standard deviation. Different letters in the same row indicate a significant difference (P < 0.05; ANOVA followed by Tukey test). Figure 2 Photomicrograph that shows myenteric ganglia in the colon stained using the Giema technique in 22-week-old male Swiss mice. TC, trained control; TI, trained infected; SC, sedentary control; SI, sedentary infected. Infection occurred with 1300 blood trypomastigotes of the Y strain of T. cruzi for 75 days. A photonic microscope (MOTIC B5) with a 2009 objective was used (ANOVA followed by Tukey test). Table 3 Morphometry of myenteric neurons of the colon in 22-week-old male Swiss mice. Infection occurred with 1300 blood trypomastigotes of the Y strain of T. cruzi for 75 days Group Parameter TC (n = 5) TI (n = 5) SC (n = 5) SI (n = 5) Nucleus area (lm 2 ) 53.5 a (40.8; 69.6) 69.6 b (54.3; 89.4) 45.8 c (34.4; 60.0) 58.3 d (44.2; 76.6) Cytoplasm area (lm 2 ) 75.2 a (50.0; 116.6) b (92.3; 225.7) 64.0 c (42.2; 98.8) 99.2 d (66.5; 155.2) Cell body area (lm 2 ) a (98.3; 180.7) b (160.7; 310.3) c (81.0; 155.5) d (118.0; 229.2) n, number of animals; TC, trained control; TI, trained infected; SC, sedentary control; SI, sedentary infected. The data are expressed as mean 25th and 75th percentiles. Different letters in the same row indicate a significant difference (P < 0.05; Kruskal Wallis test). caused a 27.2% increase in the nuclear area, 55.0% increase in the cytoplasm area and 40.2% increase in the neuronal body area (P < 0.05). Subtraction of the values ( ) indicated that moderate physical exercise provided a 19.5% benefit in myenteric neurons in infected animals, with a medium effect of 0.4 (Table 3).

6 Physical training 9 neurons 9 T. cruzi 431 Morphometric analysis of the intestinal wall Moderate physical exercise did not change the tunica mucosa thickness, enterocyte height or its nucleus area, the depth and width of the crypt, the submucosal tela or the tunica muscularis when comparing the TI and SI groups. However, moderate physical exercise increased the total intestinal wall thickness by 7.3% (TI 9 SI; P < 0.05). In sedentary animals, T. cruzi infection caused a significant 6.7% reduction of the total intestinal wall thickness (SI 9 SC; P < 0.05; Table 4). Number of intraepithelial lymphocytes, goblet cells and inflammatory foci Infection in trained animals caused a 44.7% increase (P > 0.05) in the number of IELs. In sedentary animals, the infection caused a 39.0% decrease (P > 0.05) in the number of IELs. Subtraction of these values ( ) indicated that moderate physical exercise provided a 5.7% benefit in infected animals, with a large effect of 1.2 (Table 5). The number of goblet cells in trained animals was significantly reduced, regardless of whether the animals were infected. In the TI group, exercise was responsible for a 2.0% decrease in the number of PAS-reactive cells. This number was obtained by subtracting the comparison percentage of trained animals (TC 9 TI = 9.0%; P < 0.05) from the comparison percentage of sedentary animals (SC 9 SI = 7.1%; P < 0.05; Table 5). The reduction in the number of goblet cells caused by physical exercise was confirmed by a large effect of 5.4 (TI 9 SI). Similar results were observed for sialomucins and sulphomucins (AB ph 2.5+) and reactive sulphomucins (AB ph 1.0+). Moderate physical exercise was responsible for reductions of 15.5% (TC 9 TI = 20.0%; SC 9 SI = 2.5%; P < 0.05) and 18.0% (TC 9 IT = 18.0%; SC 9 SI = 0.0%; P < 0.05; Table 5), which was confirmed by large effects of 4.0 and 3.0 (TI 9 SI). The number of inflammatory foci in the intestinal tunica muscularis in the TI group was 64.5% lower than in the SI group (P < 0.05). Moderate physical exercise was responsible for 72.2% prevention (TI TC 9 100/SI SC) of the formation of inflammatory foci (Figures 3,4), confirmed by a large effect of 1.8 (TI 9 SI). Cytokine doses Figure 5a shows the comparisons of TNF-a between groups. Moderate physical exercise caused a significant 18.0% increase (P < 0.05) in TNF-a synthesis. These data were Table 4 Morphometric analysis of the colon intestinal wall in 22-week-old male Swiss mice. Infection occurred with 1300 blood trypomastigotes of the Y strain of T. cruzi for 75 days Measurements TC (n = 5) TI (n = 5) SC (n = 5) SI (n = 5) Tunica mucosa (lm) a (126.3; 181.0) a (133.2; 179.2) b (124.3; 174.1) a (131.3; 179.1) Enterocyte height (lm) 24.2 a (23.1; 25.3) 23.1 b (22.4; 25.4) 23.4 b (22.4; 25.3) 23.2 b (22.1; 25.1) Enterocyte nucleus area (lm 2 ) 23.2 a (21.4; 24.4) 23.5 a (20.3; 24.4) 23.9 b (21.3; 25.1) 23.4 a (22.9; 25.7) Depth of the crypt (lm) 89.0 a (86.3; 94.4) a (86.4; 95.5) 92.3 b (85.2; 97.3) 90.5 a (87.0; 95.3) Width of the crypt (lm) 25.5 a (23.4; 28.2) 24.4 a (23.3; 26.0) 24.3 bc (22.6; 26.3) 24.4 bac (23.3; 26.1) Submucosal tela (lm) 21.0 a (18.2; 25.2) 22.1 ba (18.0; 25.2) 23.2 bc (20.4; 26.5) 22.2 a (18.0; 24.4) Tunica muscularis (lm) a (180.3; 252.2) a (179.3; 249.2) b (204.2; 256.5) a (176.2; 244.1) Total wall (lm) a (370.2; 657.0) b (420.1; 702.0) b (420.2; 701.4) a (370.2; 624.1) n, number of animals; TC, trained control; TI, trained infected; SC, sedentary control; SI, sedentary infected. The data are expressed as mean 25th and 75th percentiles. Different letters in the same row indicate a significant difference (P < 0.05; Kruskal Wallis test). Table 5 Ratios of lymphocytes/100 epithelial cells and goblet cells/100 epithelial cells in the tunica mucosa of the colon in 22-weekold male Swiss mice. Infection occurred with 1300 blood trypomastigotes of the Y strain of T. cruzi for 75 days Lymphocytes Goblet cells Group HE PAS AB ph 1.0+ AB ph 2.5+ TC (n = 5) a a a 0.01 TI (n = 5) b b b 0.01 SC (n = 5) c c c 0.01 SI (n = 5) d c c 0.01 n, number of animals, TC, trained control; TI, trained infected; SC, sedentary control; SI, sedentary infected; HE, haematoxylin and eosin; PAS, periodic acid-schiff; AB, alcian blue. The data are expressed as mean standard deviation. Different letters in the same column indicate a significant difference (P < 0.05; ANOVA followed by Tukey test).

7 432 N. M. Moreira et al. obtained by subtracting the comparison percentage of trained animals (TC 9 TI = 23.4%; P < 0.05) from the comparison percentage of sedentary animals (SI 9 SC = 41.3%; P < 0.05), confirmed by a large effect of 1.3 (TI 9 SI). Figure 5b shows the comparisons of TGF-b between groups. The 18.0% increase in TGF-b synthesis in response to moderate physical exercise did not reach (a) (b) Figure 3 Mean and standard deviation of inflammatory foci in the muscular tunica in 22-week-old male Swiss mice. TC, trained controls (n = 5); TI, trained infected (n = 5); SC, sedentary controls (n = 5); SI, sedentary infected (n = 5). Infection occurred with 1300 blood trypomastigotes of the Y strain of T. cruzi for 75 days. (n = number of animals). *, **P < 0.05, significant difference among the four groups (ANOVA followed by Tukey test). Figure 5 Mean and standard deviation of the detected levels of (a) TNF-a and (b) TGF-b in 22-week-old male Swiss mice. TC, trained control (n = 10); TI, trained infected (n = 10); SC, sedentary control (n = 10); SI, sedentary infected (n = 10). Infection occurred with 1300 blood trypomastigotes of the Y strain of T. cruzi for 75 days. (n = number of animals). *, **P < 0.05, significant difference among the four groups (Kruskal Wallis test). Figure 4 Photomicrograph that shows inflammatory cells (arrow) in the intestinal tunica muscularis in 22-week-old male Swiss mice. TC, trained controls (n = 5); TI, trained infected (n = 5); SC, sedentary controls (n = 5); SI, sedentary infected (n = 5). Infection occurred with 1300 blood trypomastigotes of the Y strain of T. cruzi for 75 days. (n = number of animals). P < 0.05, TC 9 SC 9 TI 9 SI (ANOVA followed by Tukey test). A HE photonic microscope (MOTIC B5) with a 2009 objective was used.

8 Physical training 9 neurons 9 T. cruzi 433 statistical significance. These data were obtained by subtracting the comparison percentage of trained animals (TC 9 TI = 21.4%; P < 0.05) from the comparison percentage of sedentary animals (SI 9 SC = 39.0%; P < 0.05), with a large effect of 0.7 for this comparison. No detectable levels of IFN-c and IL-10 were found under these experimental conditions. Discussion The present study investigated the effect of moderate physical exercise on myenteric neurons and the wall of the colon in mice infected with T. cruzi by considering the parasitological and immunological aspects of infection. The parasitological parameters showed that moderate physical exercise changed the course of the infection compared with sedentarism. Peak parasitaemia and total parasitaemia were significantly lower in the TI group than in the SI group. These results are consistent with Schebeleski-Soares et al. (2009), who observed a reduction of peak parasites in trained female BALB/c mice infected with the Y strain of T. cruzi. The present study observed no significant difference in the body mass of the animals between the TI and SI groups, likely because of the balance of the immune system associated with moderate physical exercise and discontinuous treatment with benznidazole to chronicity of infection. Benznidazole, although it does not present complete healing efficacy, prevents the development of parasitaemia, which is the cause of the morbidity associated with T. cruzi infection (Ministerio da Saude 2009; Hasslocher-Moreno et al. 2012). The present data suggest that moderate physical exercise acts on regulatory mechanisms associated with the immunological control of parasitaemia. The change in the course of the infection with moderate physical exercise was reflected by significant protection of the total myenteric neuronal population in the experimental group (17.1%) and protection of the number of neurons in the total colon area (8.0%). These results indicated a large effect of exercise on the neuronal population. In a previous study, Moreira et al. (2011) found that infection with T. cruzi decreased the total myenteric neuron population by more than 50.0%, and this reduction depended on the inoculum. In the present study, moderate physical exercise decreased parasitaemia and provided neuronal protection. The animals in the TC group exhibited a significantly higher neuronal percentage than the other groups. The neuronal data obtained in the TC and SC groups are consistent with De Britto Mari et al. (2008) and Martinez Gagliardo et al. (2008), who also studied sedentary and trained rats and observed neuronal preservation in the duodenum and colon in trained rats. With regard to myenteric neuronal morphometry, animals in the TI group exhibited a significant increase in neuronal body area, which was attributable to increases in the areas of neuronal nuclei and cytoplasm, confirmed by an average effect of 0.4. This change suggests an increase in gene expression and protein production in cholinergic and nitrergic neurons, which synthesize choline acetyltransferase and neuronal nitric oxide synthase, respectively, to form acetylcholine (which is responsible for intestinal muscle contractions) and nitric oxide (which is related to intestinal muscle dilation; Furness & Costa 2006). The observed increase may have contributed to physiological adaptation and improvements in the condition of the animals at the cellular level. Moderate physical exercise appeared to improve the control of intestinal peristalsis in animals infected with T. cruzi. Chagas disease is characterized by the development of a general inflammatory process (Lana & Tafure 2011). Silveira et al. (2007) observed an increased frequency of excitatory neurons produced by substance P in the myenteric plexus of the colon in patients with Chagasic megacolon. When secreted, substance P contributes to inflammation neurogenesis (Winter et al. 1995). Silveira et al. (2007) observed high levels of substance P in patients with chagasic megacolon. This study suggests that the hypertrophy of neurons may be related to this neurochemical marker. The literature shows that capsaicin (i.e. the active component of pepper) stimulates the release of substance P. However, the continued use of this substance leads to the depletion of substance P itself (Costa 2013). Individuals with Chagas disease often do not receive adequate care in the Brazilian Health System (SUS-Unique Health System; Juberg 2009). Moderate physical exercise in conjunction with the treatment of chronic constipation in infected individuals may have a stimulatory effect on intestinal transit (Oettle 1991). The literature provides evidence that supports the hypothesis that the continuous use of capsaicin may provide benefits to Chagasic patients because it minimizes the synthesis of substance P and consequently reduces morbidity by decreasing generalized inflammation (Costa 2013). The motility disturbances associated with Chagas disease include constipation and viscera swelling with or without thickening of the intestinal wall (Silveira 2007). The present study investigated the morphology of the intestinal wall in trained animals that were subsequently infected with T. cruzi. A significant increase in total wall thickness was observed, comparing SI and TI 9 TI 9 TC respectively. The trained TI animals also exhibited a significant reduction in the proportion of goblet cells compared with the number of enterocytes. In this group, moderate physical exercise significantly reduced the number of PAS-reactive cells, with a large effect of 5.4. Exercise also reduced sialomucins (AB ph 2.5+; large effect of 4.0) and sulphomucins (AB ph 1.0+; large effect of 3.0) compared with the SI group. Mucins comprise the mucus that covers the intestinal epithelium to protect it against any aggressor (Myers et al. 2008). The present study found that the reduction of mucin secretion in the intestinal epithelium in the trained animals, regardless of whether they were infected by T. cruzi, likely occurred because moderate physical exercise inhibited the development of parasitaemia, thus preventing the formation

9 434 N. M. Moreira et al. of the inflammatory process. Thus, no need existed to increase mucin secretion, indicating that the mucus satisfactorily played its role in covering the epithelium. In sedentary and infected animals, goblet cells that secrete neutral mucins (PAS+) significantly increased secretion in response to T. cruzi infection. According to the literature (Furlan et al. 2004), goblet cells increase their rate of discharge and secretion during an infection in the intestinal mucosa. The mucin components work as false receptors for microorganisms, causing them to be surrounded by the mucus layer, thus inhibiting their pathogenic capacity and possibly making them imperceptible (Furlan et al. 2004). A significant reduction in the number of inflammatory foci was observed in the TI group compared with the SI group. Moderate physical exercise appeared to be responsible for preventing the formation of inflammatory foci, with a large effect of 1.8. This result is related to the lower number of parasites observed in trained animals. According to the literature (Silveira 2007), the vast majority of inflammatory cells in the muscle layers in Chagasic patients are mononuclear leucocytes that have cytotoxic potential. When activated by T. cruzi, these leucocytes release a toxic substance that causes the destruction of muscle cells arranged in layers, suggesting their involvement in the inflammation process and denervation induced by T. cruzi infection. The present results support the hypothesis posited by Silveira (2007). The number of inflammatory foci in the SI group was significantly increased compared with the SC group, with a large effect of infection that resulted in a significant reduction in the number of myenteric neurons (SI 9 SC). Importantly, the reduction in the number of inflammatory foci in the TI group was observed concomitant with a significant increase in the synthesis of TNF-a compared with the SI group. Similarly, moderate physical exercise significantly increased the synthesis of TNF-a in trained animals compared with sedentary animals. This increase was confirmed by a large effect of 1.3. TNF-a plays an important role in modulating the ability of the immune system to confer resistance to T. cruzi (Abbas et al. 2008). High concentrations of TNF-a also induced cachexia, which is an important predictor of body mass loss (Anker & Coats 1999). The cytokines TGF-b and IL-10 are able to regulate the immune response of the host, particularly responses that involve macrophages (Abbas et al. 2008). Animals in the TI group exhibited an 18.0% increase in the synthesis of TGFb, with a large effect of 0.7 (TI 9 SI), emphasizing the importance of moderate physical exercise in the regulation and control of the activation of immune system macrophages in response to physiological changes that result from T. cruzi infection. Necropsy fragments of the myocardium in humans with chronic Chagas disease and congestive heart failure had little expression of TGF-b, suggesting that an inhibitory factor may have acted on macrophages, which may correspond to immunodepression caused by the presence of T. cruzi (Reis et al. 2000). In conclusion, moderate physical exercise changed the course of T. cruzi infection in mice and decreased parasitaemia in the acute phase of experimental infection, in addition to protecting the total myenteric neuronal population and inducing neuronal hypertrophy. These results suggest a better prognosis for chronically infected animals with regard to the maintenance of intestinal peristalsis. Furthermore, moderate physical exercise increased the total intestinal wall thickness, increased the number of IELs, decreased the proportion of goblet cells, decreased the number of inflammatory foci and increased the synthesis of TNF-a and TGF-b. 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