Dig Dis Sci (2014) 59:307–314 DOI 10.1007/s10620-013-2901-9
ORIGINAL ARTICLE
Moderate Physical Exercise Protects Myenteric Metabolically More Active Neurons in Mice Infected with Trypanosoma cruzi Neide Martins Moreira • Solange Marta Franzo´i de Moraes • Ma´rcia Machado de Oliveira Dala´lio • Moˆnica Lu´cia Gomes • De´bora de Mello Ganc¸ales Sant’Ana • Silvana Marques de Arau´jo
Received: 17 August 2013 / Accepted: 25 September 2013 / Published online: 2 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Background Trypanosoma cruzi causes neuronal myenteric depopulation compromising intestinal function. Aim The purpose of this study was to evaluate the influence of moderate physical exercise on NADH diaphorase (NADH-d)-positive neurons in the myenteric plexus and intestinal wall of the colon in mice infected with T. cruzi. Materials and Methods Forty 30-day-old male Swiss mice were divided into the following groups: trained infected (TI), sedentary infected (SI), trained control (TC), and sedentary control. The TC and TI groups were subjected to a moderate physical exercise program on a treadmill for 8 weeks. Three days after finishing physical exercise, the TI and SI groups were intraperitoneally inoculated with 1,300 blood trypomastigotes of the Y strain of Trypanosoma cruzi. Parasitemia was evaluated from days 4 to 61 after inoculation. On day 75 of infection, myenteric neurons in the colon were quantified (NADH-d),
N. M. Moreira (&) Graduate Program in Health Sciences, State University of Maringa, Columbus Avenue, 5790, Zone 07, Maringa´, PR 87020-900, Brazil e-mail: [email protected] S. M. F. de Moraes Department of Physiological Sciences, State University of Maringa, Columbus Avenue, 5790, Zone 07, Maringa´, PR 87020-900, Brazil e-mail: [email protected] M. M. O. Dala´lio Department of Basic Health Sciences-Immunology, University of Maringa, Columbus Avenue, 5790, Zone 07, Maringa´, PR 87020-900, Brazil e-mail: [email protected]
and inflammatory foci were counted. Tumor necrosis factor-a (TNF-a) and transforming growth factor-b (TGF-b) levels were evaluated in plasma. The results were compared using analysis of variance and the Kruskal–Wallis test at a 5 % significance level. Results Moderate physical exercise reduced the parasite peak on day 8 of infection (p = 0.0132) and total parasitemia (p = 0.0307). It also prevented neuronal depopulation (p \ 0.01), caused hypertrophy of these cells (p \ 0.05), prevented the formation of inflammatory foci (p \ 0.01), and increased the synthesis of TNF-a (p \ 0.01) and TGF-b (p [ 0.05). Conclusion These results reinforce the therapeutic benefits of moderate physical exercise for T. cruzi infection. Keywords Physical exercise Trypanosoma cruzi NADH-diaphorase positive neurons Myenteric plexus
M. L. Gomes S. M. de Arau´jo (&) Department of Immunology-Parasitology, University of Maringa, Columbus Avenue, 5790, Zone 07, Maringa´, PR 87020-900, Brazil e-mail: [email protected] M. L. Gomes e-mail: [email protected] D. M. G. Sant’Ana Graduate Program in Applied Biosciences Pharmacy, Department of Morphological Sciences, State University of Maringa, Columbus Avenue, 5790, Zone 07, Maringa´, PR 08020-900, Brazil e-mail: [email protected]
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Introduction
Materials and Methods
Trypanosoma cruzi, the etiologic agent of Chagas’ disease, affects approximately 10 million people worldwide, especially in Latin America where the disease is endemic, with more than 25 million people at risk of infection [1]. In Brazil, nearly 3 million people are infected [2]. When infecting the host, T. cruzi causes an inflammatory process [3] and activation of the immune system. This parasite then randomly invades several organs, including the heart, intestines, esophagus, and central nervous system [3]. The destruction of inhibitory and excitatory neurons in the enteric nervous system occurs in the digestive track [4–6]. More metabolically active neurons are observed among these populations [7]. Such neurons are able to maintain the homeostasis of intestinal muscle contractions and relaxation [8]. After the destruction of these neurons, muscle coordination decreases, and peristaltic movement is altered [3]. Individuals with these changes exhibit the retention of food bolus or fecal bolus, with consequent dilatation of the organ [9]. Further studies on more metabolically active neurons in the myenteric plexus in animals infected with T. cruzi are necessary. Benznidazole and nifurtimox are currently available for the treatment of Chagas’ disease. However, both of these drugs can cause serious side effects and are less effective in the chronic phase of the disease [10]. Even treated patients may experience symptoms of constipation and severe intestinal complications [11]. Therefore, the search for alternative or complementary treatments has been a great challenge [12]. Infection by T. cruzi promotes significant destruction of myenteric neurons [4, 5]. Moderate physical exercise represents a resistance factor against the development of infection caused by T. cruzi in animals [13–15], modulating immune responses and neuroimunoendocrina [14, 16, 17]. Research with animals, where it is possible to control the load of exercise and the presence of infectious agents, shows the benefits of this practice [13–15]. Specifically, moderate physical exercise promotes effective protection of the total neuronal population in mice infected with T. cruzi infection [18]. Comparing healthy sedentary and trained mice, the practice of moderate exercise had a protective effect for more metabolically active neurons and nitrergic neurons [19]. However, no studies have reported changes in more metabolically active neurons in the myenteric plexus of the colon in mice subjected to physical exercise and infected with T. cruzi. The present study evaluated the influence of moderate physical exercise on NADH-diaphorase (NADH-d)-positive neurons in the myenteric plexus and colon wall in mice infected with T. cruzi and considered parasitological, immunological, and pathological aspects.
Ethical Aspects
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This study was approved by the Ethics Committee on Animal Experiments (ECAE), State University of Maringa´, Brazil (Opinion 046/2009). Animals This was a controlled, randomized, blind trial that was repeated twice. In each replicate, forty 30-day-old male Swiss mice were used and divided into two groups: trained (n = 20) and sedentary (n = 20). Moderate Physical Exercise Protocol The 30-day-old animals were subjected to a moderate aerobic physical exercise program on a treadmill (Inbrasport ClassicCI model, Maringa, Brazil) for 8 weeks, which consisted of a daily training session five times per week for 30–45 min at a speed of 6–14 m/min in the first week, 45–60 min at a speed of 8–16 m/min in the second week, and 60 min at a speed of 10–20 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 subsequent weeks. The training sessions began at 6:00 p.m. during the animal’s dark cycle (i.e., active phase). This protocol requires light or mild effort [14, 20]. Shock or similar mechanisms were not used to induce the animal to exercise. Infection After finishing the physical exercise program, the animals were redistributed into the following groups: trained control (TC; n = 10), trained infected (TI; n = 10), sedentary control (SC; n = 10), and sedentary infected (SI; n = 10). The animals in the TI and SI groups were then intraperitoneally inoculated with 1,300 bloodstream forms of the Y strain of T. cruzi 3 days after the end of the exercise program. The chronic phase was induced with three doses of benznidazole (LAFEPE, PE, Brazil; 100 mg/kg/bm) 11, 15, and 22 days after inoculation and two doses of benznidazole (250 mg/kg/bm) 18 and 41 days after inoculation by oral gavage. Evaluation of the Course of the Infection Parasitemia was evaluated in all of the infected animals using Brener’s technique [21]. Parasite scores were calculated daily from infection days 4–15 and then on alternate days from infection days 15–61. The curve was traced using the mean parasitemia score of the animals. Based on
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the analysis of the parasitemia curve, the pre-patent and patent parasitemia periods were defined, in addition to the parasite peak and total parasitemia. The percentage of mortality of the animals was calculated, and body mass was measured weekly (in grams). Euthanasia and Organ Collection On infection day 75, the animals were sacrificed. The colon of five animals from each group was subjected to NADH-d histochemistry according to Gabella [7]. The colon area (in cm2) was calculated by measuring the width and length of the collected material. Quantitative Analysis of NADH-d Myenteric Neurons The neurons in 120 microscopic fields distributed throughout the intestinal circumference were quantified. A photonic microscope (Olympus CBA) was used to evaluate 45.6 mm2 area microscopic fields using a 409 objective. Denervation was evaluated by comparing the test and control groups and is expressed as a percentage. Morphometric Analysis of NADH-d Myenteric Neurons The area (lm2) of the cell body and nuclei of 300 neurons in the myenteric plexus distributed throughout the intestinal circumference was measured for each animal. The images were acquired by a trinocular photonic microscope (MOTIC B5) coupled to a high-definition digital video camera (MOTICAM 2000). The measurements were performed using Image-Pro Plus software. Histological and Quantitative Analysis of Inflammatory Foci in the Intestinal Wall Three 4-lm-thick histological cross sections from five animals of each group were used to measure the inflammatory foci and number of inflammatory cells in ten microscopic fields (1.9 mm2 diameter) distributed throughout the intestinal circumference and in the muscular tunica of three histologic sections from each animal to obtain the average. Foci with ten or more inflammatory cells were considered inflammatory [22]. Hematoxylineosin staining was performed, and a photonic microscope (Olympus CBA) with a 409 objective was used. Cytokine Dose Interleukin-10 (IL-10), interferon-c (IFN-c), tumor necrosis factor-a (TNF-a), and transforming growth factor-b (TGF-b) were dosed. The plasm of ten animals per group
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was used, and the enzyme-linked immunosorbent assay capture technique was applied using antibody pairs from R&D Systems (Minneapolis, MN, USA). The technique was developed according to the protocol provided by the manufacturer. The concentration of the cytokines was 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 ng/mL for TGF-b. Statistical Analysis The data distribution was verified by applying the D’Agostino Pearson or Shapiro–Wilk test. Data with a normal distribution are expressed as mean ± standard deviation 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 (P25 and P75) and compared using the Kruskal–Wallis test and median test. A 5 % significance level was adopted. The data were analyzed using BioEstat 5.0 software. The statistical effect (http://www.uccs.edu/ *lbecker/; accessed August 7, 2013) was determined using the effect size calculator test by checking the intensity of the effect of physical exercise relative to sedentarism on murine T. cruzi infection. Effect sizes B0.2 were considered small. Effect sizes B0.5 were considered medium. Effect sizes [0.5 were considered large.
Results Course of the Infection The parasite peak on day 8 of infection and total parasitemia were 48.1 % (p = 0.0077) and 72.3 % (p = 0.0065) lower, respectively, in the TI group than in the SI group. No significant difference was found between groups with regard to infectivity, the prepatent and patent periods, or mortality (p [ 0.05; Fig. 1; Table 1). During infection, the animals’ body mass remained stable in the TI and SI groups (p = 0.6914). No significant change in the length, diameter, or total area of the colon was observed (p [ 0.05). Quantitative Analysis of NADH-d Myenteric Neurons Moderate physical exercise protected NADH-d neurons by 15.2 %. These data were obtained by subtracting the comparison percentage of trained animals (TC 9 TI = 22.4 %, p \ 0.01) from the comparison percentage of sedentary animals (SI 9 SC = 37.6 %, p \ 0.01; Fig. 2; Table 2).
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statistical effect size that was small (0.02) for the TI 9 SI comparison (Table 3). Proportion of Inflammatory Foci and Cytokines
Fig. 1 Mean parasitemia 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 1,300 blood trypomastigotes of the Y strain of T. cruzi. *, **p \ 0.05, comparison between TI and SI groups on day 8 after infection (Mann–Whitney test)
The evaluation of the number of neurons in the total colon area revealed that physical exercise significantly prevented the death of NADH-d neurons by 26.0 %. These data were obtained by subtracting the comparison percentage of trained animals (TI 9 TC = 11.5 %, p \ 0.01) from the comparison percentage of sedentary animals (SI 9 SC = 37.5 %, p \ 0.01; Fig. 2; Table 2). Neuronal protection was confirmed by a statistical effect size that was large (9.1) for the TI 9 SI comparison.
The number of inflammatory foci in the intestinal tunica muscularis in the TI group was 64.5 % lower than in the SI group (p \ 0.01). Physical exercise was responsible for preventing 72.2 % (TI–TC 9 100/SI–SC) of the formation of inflammatory foci (Table 4), confirmed by a statistical effect size that was large (1.8) for the TI 9 SI comparison. Moderate physical exercise significantly increased TNFa synthesis by 18.0 % (p \ 0.01). These data were obtained by subtracting the comparison percentage of trained animals (TC 9 TI = 23.4 %, p \ 0.01) from the comparison percentage of sedentary animals (SI 9 SC = 41.3 %, p \ 0 01), confirmed by a statistical effect size that was large (1.3) for the TI 9 SI comparison (Table 4). Moderate physical exercise increased TGF-b synthesis by 17.6 %. These data were obtained by subtracting the comparison percentage of trained animals (TC 9 TI = 21.4 %, p \ 0.01) from the comparison percentage of sedentary animals (SI 9 SC = 39.0 %, p \ 0.01), confirmed by a statistical effect size that was large (0.7) for this comparison (Table 4). No detectable levels were observed for IFN-c and IL-10 under the present experimental conditions.
Discussion Morphometric Analysis of NADH-d Myenteric Neurons Moderate physical exercise and/or infection in the TI group caused hypertrophy of NADH-d neurons compared with neurons in the SI group. The data that were obtained by subtracting the values (nuclear area: TC 9 TI = 7.1 %– SC 9 SI = 11.4 %; cytoplasmic area: TC 9 TI = 6.8 %– SC 9 SI = 15.5 %; neuronal body: TC 9 TI = 24.8 %– SC 9 SI = 3.0 %) corroborated the increase and indicated the beneficial effect of physical exercise on NADH-d neurons in infected animals (nuclear area: 4.3 %; cytoplasmic area: 8.7 %; neuronal body: 21.8 %), with a
The present study determined the effect of moderate physical exercise on metabolically active neurons and the wall of the colon in mice infected with T. cruzi by considering parasitological, immunological, and pathological aspects. The parasitological data showed that moderate physical exercise differentially modulated the development of infection compared with sedentary animals. The parasite peak and total parasitemia were significantly lower in the TI group than in the SI group. These results are consistent with Schebeleski-Soares et al. [14] and Occhi et al. [15], who observed a significant reduction of the parasite peak in mice from two distinct lineages of both sexes that
Table 1 Parasitological parameters evaluated in 12-week-old male Swiss mice subjected to physical exercise and subsequently infected (TI) or maintained sedentary and subsequently infected (SI) with 1,300 blood trypomastigotes of the of Y strain of T. cruzi for 75 days Group
Infectivity (%)
Prepatent period (days)
Patent period (days)
Parasite peak (trypomastigotes/mL 9 106)
Total parasitemia (trypomastigotes/mL 9 106)
TI (n = 10)
100
5.31 ± 1.44
7.00 ± 4.21
2.98 ± 1.14
4.65 ± 1.60
SI (n = 10)
100
4.42 ± 0.51
5.58 ± 0.51
5.71a ± 3.32
16.83b ± 28.27
n = number of animals. The data are expressed as mean ± standard deviation a
p = 0.0077bp = 0.0065 (comparisons between TI and SI groups)
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Fig. 2 Photomicrograph that shows myenteric ganglia in the colon stained with NADH-d in 22-week-old male Swiss mice. TC trained control, TI trained infected, SC sedentary control, SI sedentary
infected. Infection occurred with 1,300 blood trypomastigotes of the Y strain of T. cruzi for 75 days. A photonic microscope (MOTIC B5) with a 200 9 objective was used (ANOVA followed by Tukey test)
Table 2 Neuronal myenteric density of the colon in 240 microscopic fields with an area of 45.6 mm2 in 22-week-old male Swiss mice Parameter
Group
Neuronal density in 45.6 mm2 7
Projection of the neurons number for the colon area 9 10
TC (n = 5)
TI (n = 5)
SC (n = 5)
SI (n = 5)
9,849.3a ± 756.88
7,635.2b ± 830.8
6,731.4b ± 579.9
4,195.6c ± 761.8
a
62.2 ± 45.0
b
55.0 ± 3.2
c
39.7 ± 7.4
24.8d ± 3.4
TC trained control, TI trained infected, SC sedentary control, SI sedentary infected Infection occurred with 1,300 blood trypomastigotes of the Y strain of T. cruzi for 75 days. n = number of animals. The data are expressed as mean ± standard deviation. Different letters in the same row indicate a significant difference (p \ 0.01; ANOVA followed by Tukey test)
underwent moderate exercise prior to infection with the Y strain of T. cruzi. In the present study, we did not observe a significant difference in body weight between the TI and SI groups during the course of infection (p [ 0.05). This homeostasis was likely attributable to a positive response to modulation of the immune system by moderate physical exercise and discontinuous treatment with benznidazole to chronicity of infection. Although this drug is not completely curative, it prevents the development of parasitemia, which is ultimately one of the causes of morbidity from T. cruzi infection [10]. The modulation of the progression of infection by moderate physical exercise conferred protection for more metabolically active neurons in the sample studied
(15.2 %) and the projection of the number of neurons in the total area of the colon (26.0 %). These data indicated a large effect size of physical exercise on NADH-d protection. The NADH-d histochemical technique is used to identify most metabolically active neurons by activating the enzyme NADH-d located in the mitochondrial matrix [7, 23], but this technique does not allow the differentiation of inhibitory neurons from excitatory neurons, which are responsible for dilation and constriction reflexes of the intestinal wall [6, 8]. The present results reinforce the importance of integrating a physical activity program to improve the care of individuals infected with T. cruzi in a public health context. In sedentary controls and infected animals, a decrease in the number of NADH-d neurons was found (SI 9 SC).
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Table 3 Morphometry of myenteric neurons of the colon in 22-week-old male Swiss mice. Infection occurred with 1,300 blood trypomastigotes of the Y strain of T. cruzi for 75 days Parameter
Group
Nuclei area (lm2) 2
Cytoplasm area (lm ) 2
Cell body area (lm )
TC (n = 5)
TI (n = 5)
SC (n = 5)
SI (n = 5)
70.0a (55.1; 92.0)
75.0a (57.0; 92.0)
66.4b (54.3; 87.2)
74.0ca (56.1; 93.0)
a
102.0 (72.1; 154.0) a
180.0 (133.0; 237.0)
b
96.0 (66.4; 132.3) b
173.0 (127.0; 221.0)
101.0
cb
(65.0; 140.0)
b
165.1 (135.3; 225.4)
86.0db (61.2; 147.0) 160.0b (123.2; 245.0)
TC trained control, TI trained infected, SC sedentary control, SI sedentary infected n = number of animals. 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 4 Mean and standard deviation of inflammatory foci in the tunica muscularis and concentration of TNF-a and TGF-b in 22-week-old male Swiss mice Parameter
Group TC (n = 5)
Inflammatory foci number 3
0.8a ± 0.8
TNF-a 9 10 (pg/mL)
10.7 ± 5.1
TGF-b (ng/mL)
22.4 ± 10.8
TI (n = 5) 2.8a ± 1.4 a
13.2 ± 8.8 17.6 ± 10.2
SC (n = 5)
SI (n = 5)
0.7a ± 0.9
7.9b ± 3.6
7.7 ± 4.6
4.5b ± 3.3
18.9 ± 9.3
11.6 ± 6.0
TC trained control, TI trained infected, SC sedentary control, SI sedentary infected Infection with 1,300 bloodstream forms of the Y strain of T. cruzi for 75 days. n = number of animals. 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 and Kruskall–Wallis test). Source: Moreira et al. [18]
Maifrino et al. [24] also reported a decrease in the NADH-d myenteric population in the proximal colon in mice infected with T. cruzi compared with controls. In the present study, the reduction was not related to morphological changes of the area of the colon in infected animals, whereas the length and diameter of the body remained unchanged. The animals in the TC group exhibited a significantly greater neuronal percentage than the other groups. The neuronal data obtained for the TC and SC groups are consistent with Clebis [25], in which an increase in NADHd neurons in the jejunum was observed in trained 12-month-old rats (treadmill running) compared with sedentary rats. Gagliardo [26] evaluated NADH-d neurons in the ascending and descending portions of the colon in sedentary rats (6 and 12 months of age) and rats subjected to a physical activity program (i.e., treadmill running; 12 months of age) and reported that the number of neurons was slightly increased in rats that were subjected to physical activity compared with sedentary rats. The morphometric data showed that NADH-d survivors in the TI group exhibited hypertrophy, with increases in the nuclear and cytoplasmic areas compared with the SI group (p [ 0.05), confirmed by a statistical effect size that was small (0.02). The increases in the nuclear and cytoplasmic areas of NADH-d myenteric neurons induced by moderate
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physical exercise appear to be beneficial, suggesting the increased synthesis of proteins involved in the control of contractions and relaxation of the intestinal wall. With regard to the inflammatory process in the muscle layer, a significant reduction of 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, showing an effect size that was large (1.8). This result appears to be related to the lower number of parasites observed in trained animals. According to Silveira [27], the vast majority of inflammatory cells in the muscle layers of the colon in chagasic patients, both carriers and non-carriers of megacolon, are mononuclear leukocytes, which have cytotoxic potential. When activated by T. cruzi, these leukocytes release a toxic substance that causes the destruction of muscle cell layers. The results of Silveira [27] provided a basis to hypothesize the involvement of inflammatory cells in the process of neuronal denervation induced by T. cruzi infection. The present data support the hypothesis of Silveira [27]. The number of inflammatory foci in the SI group was significantly increased compared with the SC group, with a statistical effect size that was large (2.7) for the reduction of the number NADH-d neurons (SI 9 SC).
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Notably, the reduction of the number of inflammatory foci in the TI group was observed concomitantly with increased cytokine synthesis. The animals in the TI group also produced significantly more TNF-a than the animals in the SI group. Likewise, moderate physical exercise significantly increased the synthesis of TNF-a compared with sedentary animals. This increase was confirmed by a statistical effect size that was large (1.3). The significant increase in the synthesis of TNF-a justified the reduction of the parasitemia curve, inhibiting the formation of inflammatory foci and protecting NADH-d neurons. According to the literature, high concentrations of TNFa induce cachexia, and this cytokine is an important predictor of body mass loss [28]. However, TGF-b and IL-10 are able to maintain homeostasis of the host immune response, particularly responses that involve macrophages [29]. In the present study, although not significant, animals in the TI group exhibited an increase in the synthesis of TGF-b, with a statistical effect size that was large (0.7) for the TI 9 SI comparison, emphasizing the importance of moderate physical exercise for immune control and the regulation of macrophage activation in response to physiological changes that result from T. cruzi infection in the host. Studies on fragments of human myocardium in patients with chronic Chagas’ disease and congestive heart failure, after heading died, showed low expression of TGFb, suggesting that an inhibitory factor acts on macrophages and may correspond to the immunosuppression caused by the presence of T. cruzi [30]. In conclusion, moderate physical exercise limited the development of infection by decreasing parasitemia in the acute phase in mice inoculated with T. cruzi, protected the population of more metabolically active neurons, and induced neuronal hypertrophy. These results suggest a better prognosis for chronically infected animals with regard to the maintenance of intestinal peristalsis. Furthermore, moderate physical exercise reduced the number of inflammatory foci and increased the synthesis of TNF-a and TGF-b. These results support the beneficial effects of moderate exercise and may contribute to the improved care of individuals chronically infected with T. cruzi. Acknowledgments The authors thank Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and the Araucaria Foundation of Parana´ for financial support. Conflict of interest
None.
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ORIGINAL ARTICLE
Moderate Physical Exercise Protects Myenteric Metabolically More Active Neurons in Mice Infected with Trypanosoma cruzi Neide Martins Moreira • Solange Marta Franzo´i de Moraes • Ma´rcia Machado de Oliveira Dala´lio • Moˆnica Lu´cia Gomes • De´bora de Mello Ganc¸ales Sant’Ana • Silvana Marques de Arau´jo
Received: 17 August 2013 / Accepted: 25 September 2013 / Published online: 2 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Background Trypanosoma cruzi causes neuronal myenteric depopulation compromising intestinal function. Aim The purpose of this study was to evaluate the influence of moderate physical exercise on NADH diaphorase (NADH-d)-positive neurons in the myenteric plexus and intestinal wall of the colon in mice infected with T. cruzi. Materials and Methods Forty 30-day-old male Swiss mice were divided into the following groups: trained infected (TI), sedentary infected (SI), trained control (TC), and sedentary control. The TC and TI groups were subjected to a moderate physical exercise program on a treadmill for 8 weeks. Three days after finishing physical exercise, the TI and SI groups were intraperitoneally inoculated with 1,300 blood trypomastigotes of the Y strain of Trypanosoma cruzi. Parasitemia was evaluated from days 4 to 61 after inoculation. On day 75 of infection, myenteric neurons in the colon were quantified (NADH-d),
N. M. Moreira (&) Graduate Program in Health Sciences, State University of Maringa, Columbus Avenue, 5790, Zone 07, Maringa´, PR 87020-900, Brazil e-mail: [email protected] S. M. F. de Moraes Department of Physiological Sciences, State University of Maringa, Columbus Avenue, 5790, Zone 07, Maringa´, PR 87020-900, Brazil e-mail: [email protected] M. M. O. Dala´lio Department of Basic Health Sciences-Immunology, University of Maringa, Columbus Avenue, 5790, Zone 07, Maringa´, PR 87020-900, Brazil e-mail: [email protected]
and inflammatory foci were counted. Tumor necrosis factor-a (TNF-a) and transforming growth factor-b (TGF-b) levels were evaluated in plasma. The results were compared using analysis of variance and the Kruskal–Wallis test at a 5 % significance level. Results Moderate physical exercise reduced the parasite peak on day 8 of infection (p = 0.0132) and total parasitemia (p = 0.0307). It also prevented neuronal depopulation (p \ 0.01), caused hypertrophy of these cells (p \ 0.05), prevented the formation of inflammatory foci (p \ 0.01), and increased the synthesis of TNF-a (p \ 0.01) and TGF-b (p [ 0.05). Conclusion These results reinforce the therapeutic benefits of moderate physical exercise for T. cruzi infection. Keywords Physical exercise Trypanosoma cruzi NADH-diaphorase positive neurons Myenteric plexus
M. L. Gomes S. M. de Arau´jo (&) Department of Immunology-Parasitology, University of Maringa, Columbus Avenue, 5790, Zone 07, Maringa´, PR 87020-900, Brazil e-mail: [email protected] M. L. Gomes e-mail: [email protected] D. M. G. Sant’Ana Graduate Program in Applied Biosciences Pharmacy, Department of Morphological Sciences, State University of Maringa, Columbus Avenue, 5790, Zone 07, Maringa´, PR 08020-900, Brazil e-mail: [email protected]
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Introduction
Materials and Methods
Trypanosoma cruzi, the etiologic agent of Chagas’ disease, affects approximately 10 million people worldwide, especially in Latin America where the disease is endemic, with more than 25 million people at risk of infection [1]. In Brazil, nearly 3 million people are infected [2]. When infecting the host, T. cruzi causes an inflammatory process [3] and activation of the immune system. This parasite then randomly invades several organs, including the heart, intestines, esophagus, and central nervous system [3]. The destruction of inhibitory and excitatory neurons in the enteric nervous system occurs in the digestive track [4–6]. More metabolically active neurons are observed among these populations [7]. Such neurons are able to maintain the homeostasis of intestinal muscle contractions and relaxation [8]. After the destruction of these neurons, muscle coordination decreases, and peristaltic movement is altered [3]. Individuals with these changes exhibit the retention of food bolus or fecal bolus, with consequent dilatation of the organ [9]. Further studies on more metabolically active neurons in the myenteric plexus in animals infected with T. cruzi are necessary. Benznidazole and nifurtimox are currently available for the treatment of Chagas’ disease. However, both of these drugs can cause serious side effects and are less effective in the chronic phase of the disease [10]. Even treated patients may experience symptoms of constipation and severe intestinal complications [11]. Therefore, the search for alternative or complementary treatments has been a great challenge [12]. Infection by T. cruzi promotes significant destruction of myenteric neurons [4, 5]. Moderate physical exercise represents a resistance factor against the development of infection caused by T. cruzi in animals [13–15], modulating immune responses and neuroimunoendocrina [14, 16, 17]. Research with animals, where it is possible to control the load of exercise and the presence of infectious agents, shows the benefits of this practice [13–15]. Specifically, moderate physical exercise promotes effective protection of the total neuronal population in mice infected with T. cruzi infection [18]. Comparing healthy sedentary and trained mice, the practice of moderate exercise had a protective effect for more metabolically active neurons and nitrergic neurons [19]. However, no studies have reported changes in more metabolically active neurons in the myenteric plexus of the colon in mice subjected to physical exercise and infected with T. cruzi. The present study evaluated the influence of moderate physical exercise on NADH-diaphorase (NADH-d)-positive neurons in the myenteric plexus and colon wall in mice infected with T. cruzi and considered parasitological, immunological, and pathological aspects.
Ethical Aspects
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This study was approved by the Ethics Committee on Animal Experiments (ECAE), State University of Maringa´, Brazil (Opinion 046/2009). Animals This was a controlled, randomized, blind trial that was repeated twice. In each replicate, forty 30-day-old male Swiss mice were used and divided into two groups: trained (n = 20) and sedentary (n = 20). Moderate Physical Exercise Protocol The 30-day-old animals were subjected to a moderate aerobic physical exercise program on a treadmill (Inbrasport ClassicCI model, Maringa, Brazil) for 8 weeks, which consisted of a daily training session five times per week for 30–45 min at a speed of 6–14 m/min in the first week, 45–60 min at a speed of 8–16 m/min in the second week, and 60 min at a speed of 10–20 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 subsequent weeks. The training sessions began at 6:00 p.m. during the animal’s dark cycle (i.e., active phase). This protocol requires light or mild effort [14, 20]. Shock or similar mechanisms were not used to induce the animal to exercise. Infection After finishing the physical exercise program, the animals were redistributed into the following groups: trained control (TC; n = 10), trained infected (TI; n = 10), sedentary control (SC; n = 10), and sedentary infected (SI; n = 10). The animals in the TI and SI groups were then intraperitoneally inoculated with 1,300 bloodstream forms of the Y strain of T. cruzi 3 days after the end of the exercise program. The chronic phase was induced with three doses of benznidazole (LAFEPE, PE, Brazil; 100 mg/kg/bm) 11, 15, and 22 days after inoculation and two doses of benznidazole (250 mg/kg/bm) 18 and 41 days after inoculation by oral gavage. Evaluation of the Course of the Infection Parasitemia was evaluated in all of the infected animals using Brener’s technique [21]. Parasite scores were calculated daily from infection days 4–15 and then on alternate days from infection days 15–61. The curve was traced using the mean parasitemia score of the animals. Based on
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the analysis of the parasitemia curve, the pre-patent and patent parasitemia periods were defined, in addition to the parasite peak and total parasitemia. The percentage of mortality of the animals was calculated, and body mass was measured weekly (in grams). Euthanasia and Organ Collection On infection day 75, the animals were sacrificed. The colon of five animals from each group was subjected to NADH-d histochemistry according to Gabella [7]. The colon area (in cm2) was calculated by measuring the width and length of the collected material. Quantitative Analysis of NADH-d Myenteric Neurons The neurons in 120 microscopic fields distributed throughout the intestinal circumference were quantified. A photonic microscope (Olympus CBA) was used to evaluate 45.6 mm2 area microscopic fields using a 409 objective. Denervation was evaluated by comparing the test and control groups and is expressed as a percentage. Morphometric Analysis of NADH-d Myenteric Neurons The area (lm2) of the cell body and nuclei of 300 neurons in the myenteric plexus distributed throughout the intestinal circumference was measured for each animal. The images were acquired by a trinocular photonic microscope (MOTIC B5) coupled to a high-definition digital video camera (MOTICAM 2000). The measurements were performed using Image-Pro Plus software. Histological and Quantitative Analysis of Inflammatory Foci in the Intestinal Wall Three 4-lm-thick histological cross sections from five animals of each group were used to measure the inflammatory foci and number of inflammatory cells in ten microscopic fields (1.9 mm2 diameter) distributed throughout the intestinal circumference and in the muscular tunica of three histologic sections from each animal to obtain the average. Foci with ten or more inflammatory cells were considered inflammatory [22]. Hematoxylineosin staining was performed, and a photonic microscope (Olympus CBA) with a 409 objective was used. Cytokine Dose Interleukin-10 (IL-10), interferon-c (IFN-c), tumor necrosis factor-a (TNF-a), and transforming growth factor-b (TGF-b) were dosed. The plasm of ten animals per group
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was used, and the enzyme-linked immunosorbent assay capture technique was applied using antibody pairs from R&D Systems (Minneapolis, MN, USA). The technique was developed according to the protocol provided by the manufacturer. The concentration of the cytokines was 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 ng/mL for TGF-b. Statistical Analysis The data distribution was verified by applying the D’Agostino Pearson or Shapiro–Wilk test. Data with a normal distribution are expressed as mean ± standard deviation 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 (P25 and P75) and compared using the Kruskal–Wallis test and median test. A 5 % significance level was adopted. The data were analyzed using BioEstat 5.0 software. The statistical effect (http://www.uccs.edu/ *lbecker/; accessed August 7, 2013) was determined using the effect size calculator test by checking the intensity of the effect of physical exercise relative to sedentarism on murine T. cruzi infection. Effect sizes B0.2 were considered small. Effect sizes B0.5 were considered medium. Effect sizes [0.5 were considered large.
Results Course of the Infection The parasite peak on day 8 of infection and total parasitemia were 48.1 % (p = 0.0077) and 72.3 % (p = 0.0065) lower, respectively, in the TI group than in the SI group. No significant difference was found between groups with regard to infectivity, the prepatent and patent periods, or mortality (p [ 0.05; Fig. 1; Table 1). During infection, the animals’ body mass remained stable in the TI and SI groups (p = 0.6914). No significant change in the length, diameter, or total area of the colon was observed (p [ 0.05). Quantitative Analysis of NADH-d Myenteric Neurons Moderate physical exercise protected NADH-d neurons by 15.2 %. These data were obtained by subtracting the comparison percentage of trained animals (TC 9 TI = 22.4 %, p \ 0.01) from the comparison percentage of sedentary animals (SI 9 SC = 37.6 %, p \ 0.01; Fig. 2; Table 2).
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statistical effect size that was small (0.02) for the TI 9 SI comparison (Table 3). Proportion of Inflammatory Foci and Cytokines
Fig. 1 Mean parasitemia 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 1,300 blood trypomastigotes of the Y strain of T. cruzi. *, **p \ 0.05, comparison between TI and SI groups on day 8 after infection (Mann–Whitney test)
The evaluation of the number of neurons in the total colon area revealed that physical exercise significantly prevented the death of NADH-d neurons by 26.0 %. These data were obtained by subtracting the comparison percentage of trained animals (TI 9 TC = 11.5 %, p \ 0.01) from the comparison percentage of sedentary animals (SI 9 SC = 37.5 %, p \ 0.01; Fig. 2; Table 2). Neuronal protection was confirmed by a statistical effect size that was large (9.1) for the TI 9 SI comparison.
The number of inflammatory foci in the intestinal tunica muscularis in the TI group was 64.5 % lower than in the SI group (p \ 0.01). Physical exercise was responsible for preventing 72.2 % (TI–TC 9 100/SI–SC) of the formation of inflammatory foci (Table 4), confirmed by a statistical effect size that was large (1.8) for the TI 9 SI comparison. Moderate physical exercise significantly increased TNFa synthesis by 18.0 % (p \ 0.01). These data were obtained by subtracting the comparison percentage of trained animals (TC 9 TI = 23.4 %, p \ 0.01) from the comparison percentage of sedentary animals (SI 9 SC = 41.3 %, p \ 0 01), confirmed by a statistical effect size that was large (1.3) for the TI 9 SI comparison (Table 4). Moderate physical exercise increased TGF-b synthesis by 17.6 %. These data were obtained by subtracting the comparison percentage of trained animals (TC 9 TI = 21.4 %, p \ 0.01) from the comparison percentage of sedentary animals (SI 9 SC = 39.0 %, p \ 0.01), confirmed by a statistical effect size that was large (0.7) for this comparison (Table 4). No detectable levels were observed for IFN-c and IL-10 under the present experimental conditions.
Discussion Morphometric Analysis of NADH-d Myenteric Neurons Moderate physical exercise and/or infection in the TI group caused hypertrophy of NADH-d neurons compared with neurons in the SI group. The data that were obtained by subtracting the values (nuclear area: TC 9 TI = 7.1 %– SC 9 SI = 11.4 %; cytoplasmic area: TC 9 TI = 6.8 %– SC 9 SI = 15.5 %; neuronal body: TC 9 TI = 24.8 %– SC 9 SI = 3.0 %) corroborated the increase and indicated the beneficial effect of physical exercise on NADH-d neurons in infected animals (nuclear area: 4.3 %; cytoplasmic area: 8.7 %; neuronal body: 21.8 %), with a
The present study determined the effect of moderate physical exercise on metabolically active neurons and the wall of the colon in mice infected with T. cruzi by considering parasitological, immunological, and pathological aspects. The parasitological data showed that moderate physical exercise differentially modulated the development of infection compared with sedentary animals. The parasite peak and total parasitemia were significantly lower in the TI group than in the SI group. These results are consistent with Schebeleski-Soares et al. [14] and Occhi et al. [15], who observed a significant reduction of the parasite peak in mice from two distinct lineages of both sexes that
Table 1 Parasitological parameters evaluated in 12-week-old male Swiss mice subjected to physical exercise and subsequently infected (TI) or maintained sedentary and subsequently infected (SI) with 1,300 blood trypomastigotes of the of Y strain of T. cruzi for 75 days Group
Infectivity (%)
Prepatent period (days)
Patent period (days)
Parasite peak (trypomastigotes/mL 9 106)
Total parasitemia (trypomastigotes/mL 9 106)
TI (n = 10)
100
5.31 ± 1.44
7.00 ± 4.21
2.98 ± 1.14
4.65 ± 1.60
SI (n = 10)
100
4.42 ± 0.51
5.58 ± 0.51
5.71a ± 3.32
16.83b ± 28.27
n = number of animals. The data are expressed as mean ± standard deviation a
p = 0.0077bp = 0.0065 (comparisons between TI and SI groups)
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311
Fig. 2 Photomicrograph that shows myenteric ganglia in the colon stained with NADH-d in 22-week-old male Swiss mice. TC trained control, TI trained infected, SC sedentary control, SI sedentary
infected. Infection occurred with 1,300 blood trypomastigotes of the Y strain of T. cruzi for 75 days. A photonic microscope (MOTIC B5) with a 200 9 objective was used (ANOVA followed by Tukey test)
Table 2 Neuronal myenteric density of the colon in 240 microscopic fields with an area of 45.6 mm2 in 22-week-old male Swiss mice Parameter
Group
Neuronal density in 45.6 mm2 7
Projection of the neurons number for the colon area 9 10
TC (n = 5)
TI (n = 5)
SC (n = 5)
SI (n = 5)
9,849.3a ± 756.88
7,635.2b ± 830.8
6,731.4b ± 579.9
4,195.6c ± 761.8
a
62.2 ± 45.0
b
55.0 ± 3.2
c
39.7 ± 7.4
24.8d ± 3.4
TC trained control, TI trained infected, SC sedentary control, SI sedentary infected Infection occurred with 1,300 blood trypomastigotes of the Y strain of T. cruzi for 75 days. n = number of animals. The data are expressed as mean ± standard deviation. Different letters in the same row indicate a significant difference (p \ 0.01; ANOVA followed by Tukey test)
underwent moderate exercise prior to infection with the Y strain of T. cruzi. In the present study, we did not observe a significant difference in body weight between the TI and SI groups during the course of infection (p [ 0.05). This homeostasis was likely attributable to a positive response to modulation of the immune system by moderate physical exercise and discontinuous treatment with benznidazole to chronicity of infection. Although this drug is not completely curative, it prevents the development of parasitemia, which is ultimately one of the causes of morbidity from T. cruzi infection [10]. The modulation of the progression of infection by moderate physical exercise conferred protection for more metabolically active neurons in the sample studied
(15.2 %) and the projection of the number of neurons in the total area of the colon (26.0 %). These data indicated a large effect size of physical exercise on NADH-d protection. The NADH-d histochemical technique is used to identify most metabolically active neurons by activating the enzyme NADH-d located in the mitochondrial matrix [7, 23], but this technique does not allow the differentiation of inhibitory neurons from excitatory neurons, which are responsible for dilation and constriction reflexes of the intestinal wall [6, 8]. The present results reinforce the importance of integrating a physical activity program to improve the care of individuals infected with T. cruzi in a public health context. In sedentary controls and infected animals, a decrease in the number of NADH-d neurons was found (SI 9 SC).
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Table 3 Morphometry of myenteric neurons of the colon in 22-week-old male Swiss mice. Infection occurred with 1,300 blood trypomastigotes of the Y strain of T. cruzi for 75 days Parameter
Group
Nuclei area (lm2) 2
Cytoplasm area (lm ) 2
Cell body area (lm )
TC (n = 5)
TI (n = 5)
SC (n = 5)
SI (n = 5)
70.0a (55.1; 92.0)
75.0a (57.0; 92.0)
66.4b (54.3; 87.2)
74.0ca (56.1; 93.0)
a
102.0 (72.1; 154.0) a
180.0 (133.0; 237.0)
b
96.0 (66.4; 132.3) b
173.0 (127.0; 221.0)
101.0
cb
(65.0; 140.0)
b
165.1 (135.3; 225.4)
86.0db (61.2; 147.0) 160.0b (123.2; 245.0)
TC trained control, TI trained infected, SC sedentary control, SI sedentary infected n = number of animals. 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 4 Mean and standard deviation of inflammatory foci in the tunica muscularis and concentration of TNF-a and TGF-b in 22-week-old male Swiss mice Parameter
Group TC (n = 5)
Inflammatory foci number 3
0.8a ± 0.8
TNF-a 9 10 (pg/mL)
10.7 ± 5.1
TGF-b (ng/mL)
22.4 ± 10.8
TI (n = 5) 2.8a ± 1.4 a
13.2 ± 8.8 17.6 ± 10.2
SC (n = 5)
SI (n = 5)
0.7a ± 0.9
7.9b ± 3.6
7.7 ± 4.6
4.5b ± 3.3
18.9 ± 9.3
11.6 ± 6.0
TC trained control, TI trained infected, SC sedentary control, SI sedentary infected Infection with 1,300 bloodstream forms of the Y strain of T. cruzi for 75 days. n = number of animals. 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 and Kruskall–Wallis test). Source: Moreira et al. [18]
Maifrino et al. [24] also reported a decrease in the NADH-d myenteric population in the proximal colon in mice infected with T. cruzi compared with controls. In the present study, the reduction was not related to morphological changes of the area of the colon in infected animals, whereas the length and diameter of the body remained unchanged. The animals in the TC group exhibited a significantly greater neuronal percentage than the other groups. The neuronal data obtained for the TC and SC groups are consistent with Clebis [25], in which an increase in NADHd neurons in the jejunum was observed in trained 12-month-old rats (treadmill running) compared with sedentary rats. Gagliardo [26] evaluated NADH-d neurons in the ascending and descending portions of the colon in sedentary rats (6 and 12 months of age) and rats subjected to a physical activity program (i.e., treadmill running; 12 months of age) and reported that the number of neurons was slightly increased in rats that were subjected to physical activity compared with sedentary rats. The morphometric data showed that NADH-d survivors in the TI group exhibited hypertrophy, with increases in the nuclear and cytoplasmic areas compared with the SI group (p [ 0.05), confirmed by a statistical effect size that was small (0.02). The increases in the nuclear and cytoplasmic areas of NADH-d myenteric neurons induced by moderate
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physical exercise appear to be beneficial, suggesting the increased synthesis of proteins involved in the control of contractions and relaxation of the intestinal wall. With regard to the inflammatory process in the muscle layer, a significant reduction of 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, showing an effect size that was large (1.8). This result appears to be related to the lower number of parasites observed in trained animals. According to Silveira [27], the vast majority of inflammatory cells in the muscle layers of the colon in chagasic patients, both carriers and non-carriers of megacolon, are mononuclear leukocytes, which have cytotoxic potential. When activated by T. cruzi, these leukocytes release a toxic substance that causes the destruction of muscle cell layers. The results of Silveira [27] provided a basis to hypothesize the involvement of inflammatory cells in the process of neuronal denervation induced by T. cruzi infection. The present data support the hypothesis of Silveira [27]. The number of inflammatory foci in the SI group was significantly increased compared with the SC group, with a statistical effect size that was large (2.7) for the reduction of the number NADH-d neurons (SI 9 SC).
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Notably, the reduction of the number of inflammatory foci in the TI group was observed concomitantly with increased cytokine synthesis. The animals in the TI group also produced significantly more TNF-a than the animals in the SI group. Likewise, moderate physical exercise significantly increased the synthesis of TNF-a compared with sedentary animals. This increase was confirmed by a statistical effect size that was large (1.3). The significant increase in the synthesis of TNF-a justified the reduction of the parasitemia curve, inhibiting the formation of inflammatory foci and protecting NADH-d neurons. According to the literature, high concentrations of TNFa induce cachexia, and this cytokine is an important predictor of body mass loss [28]. However, TGF-b and IL-10 are able to maintain homeostasis of the host immune response, particularly responses that involve macrophages [29]. In the present study, although not significant, animals in the TI group exhibited an increase in the synthesis of TGF-b, with a statistical effect size that was large (0.7) for the TI 9 SI comparison, emphasizing the importance of moderate physical exercise for immune control and the regulation of macrophage activation in response to physiological changes that result from T. cruzi infection in the host. Studies on fragments of human myocardium in patients with chronic Chagas’ disease and congestive heart failure, after heading died, showed low expression of TGFb, suggesting that an inhibitory factor acts on macrophages and may correspond to the immunosuppression caused by the presence of T. cruzi [30]. In conclusion, moderate physical exercise limited the development of infection by decreasing parasitemia in the acute phase in mice inoculated with T. cruzi, protected the population of more metabolically active neurons, and induced neuronal hypertrophy. These results suggest a better prognosis for chronically infected animals with regard to the maintenance of intestinal peristalsis. Furthermore, moderate physical exercise reduced the number of inflammatory foci and increased the synthesis of TNF-a and TGF-b. These results support the beneficial effects of moderate exercise and may contribute to the improved care of individuals chronically infected with T. cruzi. Acknowledgments The authors thank Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and the Araucaria Foundation of Parana´ for financial support. Conflict of interest
None.
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