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Regenerative process evaluation of neuronal subclasses in chagasic patients with megacolon
Human Immunology 74 (2013) 181–188
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Regenerative process evaluation of neuronal subclasses in chagasic patients with megacolon Milena Dionízio Moreira a, Axel Brehmer b, Enio Chaves de Oliveira c, Salustiano Gabriel Neto c, Alejandro O. Luquetti c, Lilian Lacerda Bueno d, Ricardo Toshio Fujiwara d, Michelle Aparecida Ribeiro de Freitas e, Alexandre Barcelos Morais da Silveira a,⇑ a
Neurosciences Laboratory, Human Anatomy Department, ICBIM, Universidade Federal de Uberlândia, Minas Gerais 38.400-902, Brazil Institute of Anatomy I, University of Erlangen–Nuremberg, Krankenhausstr. 9, Erlangen D-91054, Germany Department of Surgery, Medical School, Universidade Federal de Goiás, Goiânia, Goiás 74.605-020, Brazil d Department of Parasitology, ICB, Universidade Federal de Minas Gerais 31.270-901, Brazil e Parasitology Department, ICBIM, Universidade Federal de Uberlândia, Minas Gerais 38.400-902, Brazil b c
a r t i c l e
i n f o
Article history: Received 27 June 2012 Accepted 27 November 2012 Available online 5 December 2012
a b s t r a c t Chagas’ disease is one of the most serious parasitic diseases of Latin America, with a social and economic impact far outweighing the combined effects of other parasitic diseases such as malaria, leishmaniasis and schistosomiasis. In the chronic phase of this disease, the destruction of enteric nervous system (ENS) components leads to megacolon development. Previous data presented that the regeneration tax in the ENS neurons is augmented in chagasic patients. Although, there are several neuronal types with different functions in the intestine a detailed study about the regeneration of every neuronal type was never performed before. Therefore, the aim of this study was to evaluate the regeneration tax of every neuronal cell type in the ENS from chagasic patients with megacolon and non-infected individuals. A neuronal regeneration marker (GAP-43) was used in combination with a pan-neuronal marker (Peripherin) and several neuropeptides markers (cChat, Substance P, NPY, VIP and NOS), and it was considered as positive just with the combination of these markers. Our results demonstrated that the regeneration levels of cChat, Substance P, and NPY were similar in chagasic patients and non-infected individuals. However, levels of VIP and NOS neuropeptides were increased in chagasic patients when compared with noninfected individuals. We believe that the augment in the regeneration occur due to an increased destruction of selective neuronal types. These results corroborates with previous studies that pointed out to selective destruction of VIP and NOS neurons in chagasic patients. Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Chagas’ disease, caused by the Trypanosoma cruzi parasite, was discovered in 1909 by researcher Carlos Chagas, in Lassance, a city in the state of Minas Gerais, Brazil. It is estimated that currently 8 million people present the disease throughout the Americas, and is related to environmental and socio-political issues such as education, housing and migration [1,2]. In the last century, the main form of transmission of Chagas’ disease was by the hematophagous triatomine insect known as ‘‘kissing bug’’ [3]. In the last decades, aside from triatomine transmission, the parasite has also been transmitted via blood transfusions, organ transplants, via
⇑ Corresponding author. Address: Neurosciences Laboratory, Human Anatomy Sector, ICBIM, Campus Umuarama, Universidade Federal de Uberlândia, Minas Gerais 38400-902, Brazil. Fax: +55 34 3218 2472. E-mail address: [email protected] (A.B.M. da Silveira).
placenta and even via the consumption of foods contaminated by the parasite [4–6]. This disease presents two well defined phases: the acute phase and the chronic phase. The acute phase, lasting approximately two to three months, is characterized by an abundance of circulating parasites, fever, asthenia and malaise. After this phase, the individual goes into an asymptomatic state, which characterizes the beginning of the chronic phase. Years after the acute phase, the patient can continue not exhibiting symptoms, characterizing the indeterminate form of the disease, or can even develop pathological alterations in the functioning of the heart (cardiac form) and present gastrointestinal manifestations, culminating in the digestive form of the disease [1,3,7]. The digestive form of Chagas’ disease is manifested mainly as the megaesophagus and megacolon [1,8]. The megacolon is characterized by intestinal dilation associated with several focus of inflammatory process. Previous studies have shown that these focuses are made up of T CD3 lymphocytes, B CD20 lymphocytes and
0198-8859/$36.00 - see front matter Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humimm.2012.11.012
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natural killer cells and cytotoxic T lymphocytes. Nowadays, it is accepted that this inflammatory process is, at least in part, responsible for the destruction of components of the enteric nervous system [9–11]. The enteric nervous system is a division of the autonomous nervous system and represents the intrinsic innervation of the gastrointestinal tract. It is made up of neurons and cells incorporated in the intestinal wall. These neurons are located in the submucosa (submucosal plexus) and between the external and internal muscle layers (myenteric plexus), interconnected by bundles of neuronal filaments [12–15]. The enteric neurons can be identified by their function, morphology and neurochemical correlation. Functionally, they can be divided into: excitatory motor neurons, inhibitory motor neurons, interneurons and intrinsic primary afferent neurons (IPANs) [15]. The excitatory motor neurons innervate the longitudinal and circular smooth muscle of the entire digestive tract. The main transmitter of these neurons is acetylcholine (ACh) which acts in the muscle through muscarinic receptors. Tachykinins represented by the substance P (SP), contribute to the excitatory transmission, but have a secondary role with compared to that of ACh [15]. The inhibitory motor neurons release a combination of transmitters that contribute to the relaxation of the gastrointestinal tract. The primary neurotransmitter of these neurons is nitric oxide (NO) which receives the secondary contribution of other substances such as vasoactive intestinal peptide (VIP) and adenosine triphosphate (ATP). The interneurons form chains that run along the intestine and can be identified in all regions. This neuronal class has somastostatin and neuropeptide Y (NPY) as markers and, in some cases, serotonin (5-HT) [13,15]. The intrinsic primary afferent neurons (IPAN) are numerous, approximately 500 per millimeter and, by the immunohistochemical technique, its best marker is calretinin. They are directly sensitive to the mechanical and chemical stimuli of the intestinal mucosa and the sum of synaptic events caused by the transmission of IPAN result in the activation of numerous interneurons and motor neurons [13,16,17]. When these neurons suffer an aggression, they can present a process of regeneration known as neuronal plasticity. This process can be identified through a neuronal differentiation marker called growth associated protein-43 (GAP-43). GAP-43 is an integral membrane protein, existing in elevated levels when there is development or regeneration of neurons [18–21]. The characterization of SNE alterations in Chagas’ disease has been greatly emphasized in the last few years [11,17,19,22–26]. Previous results in our research group demonstrated that there is a significant rate of regeneration in patients that did not develop the megacolon. However, it remains to be seen whether this neuronal regeneration occurs in all neuronal subclasses or if it is restricted to some neuronal groups. Thus, the objective of this work is to characterize the intensity of neuronal regeneration in the subclasses of neurons of the SNE in patients with Chagas’ disease. We believe these results may contribute not only to partially elucidate the pathogenesis of Chagas’ disease, but also to the understanding of the physiological functioning of the gastrointestinal tract.
2. Material and methods 2.1. Patients Tissue samples from chagasic patients with megacolon and a non-infected group used in this study, were collected via surgery or necropsy at Medical School Hospital - Federal University of Goiás, by Dr. Enio Chaves Oliveira. Dilatation was diagnosed with plain abdominal X-ray, digital rectal examination, and barium enema. Previous consent was given by all individuals, parents or
legal guardians for inclusion in the research. The use of these samples for scientific purposes was previously authorized and approved by the Research Ethic Committee of the Universidade Federal de Uberlândia (No. 110/11). Patient data is summarized in Table 1. Samples were collected only from dilated segment, with at least 4 cm of length and variable tissue circumference according to the amount of dilatation. The samples were rinsed with phosphate buffered saline (pH 7.2–7.4) and further fixated. During fixation, the gut samples were distended with fixative solution (freshly diluted solution of 4% paraformaldehyde in phosphate buffered saline – pH 7.2–7.4) using a syringe. Then, the inflated segments were placed in fixative solution for 2–4 h. After this time, the inflated segment was opened, separated in small pieces and stored in 0.05% thimerosal in 0.1 M phosphate buffered saline (pH 7.2–7.4) at 4 °C. Alternately, short segments were pinned out in a Sylgard lined Petri dish and transferred to 4% formalin in 0.1 M phosphate buffer (pH 7.4) at room temperature for 2–3 h. After several washes in 0.05 M TRIS-buffered saline (TBS; pH 7.4), three longitudinal muscle/myenteric plexus wholemounts (2 cm length, 1 cm width) per segment (derived from each patient) were prepared. In the following day, the small segments of tissue were
Table 1 Patients data. Groups
Gender
Age
Megacolon
Diagnostic
Non-infected group
M
46
No
M
75
No
F
75
No
F
72
No
F
58
No
F
58
No
F
43
No
F
81
No
Rectal adenocarcinoma Sigmoid adenocarcinoma Sigmoid diverticular disease Sigmoid diverticular disease Sigmoid diverticular disease Sigmoid adenocarcinoma Sigmoid diverticular disease Sigmoid adenocarcinoma
F M M F M F F F M F F F F
57 62 60 62 58 76 43 69 59 61 53 48 58
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Chagasic patients with megacolon
Megacolon Megacolon Megacolon Megacolon Megacolon Megacolon Megacolon Megacolon Megacolon Megacolon Megacolon Megacolon Megacolon
Table 2 Primary antibodies. Antibody
Source
Code
Dilution
Anti-peripherin Anti-calretinin Anti-substanciap Anti-cChAT Anti-neuropeptideo Y Anti-NOS Anti-VIP Anti-Gap-43
INVITROGEN DAKO INVITROGEN Advanced targeting systems SIGMA SIGMA SIGMA SIGMA
A-21272 M7245 180091 AB-N34 N9528 N7280 V3508 G8043
1:1000 1:200 1:1000 1:500 1:200 1:100 1:500 1:500
M.D. Moreira et al. / Human Immunology 74 (2013) 181–188 Table 3 Secondary antibodies. Antibody
Company
Dilution (ll)
Donkey anti-goat ALEXA 488 Donkey anti-mouse ALEXA 555 Donkey anti-rabbit ALEXA 488
Molecular Probes Molecular Probes Molecular Probes
1:1000 1:1000 1:1000
transferred to a mixture of PBS-sucrose-azide and OCT compound (Tissue Tek, Elkhart, IN, USA) at a ratio of 1:1 for 24 h before being embedded in 100% OCT. Sections 12 lm thick were cut and mounted on microscope slides and dried for 1 h at room temperature. 2.2. Immunohistochemistry The wholemounts were pre-incubated for 2 h in TBS 0.05 M (pH 7.4) with 1% bovine serum albumin (BSA), 0.5% Triton X-100, 0.05% thimerosal and 5% goat serum. After being rinsed in TBS for 10 min, they were incubated in a solution containing BSA, Triton X-100, thimerosal and the primary antibodies (Table 2) for 24 h at 4 °C.
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In the next day, the wholemounts were washed in TBS and then the secondary antibodies (Table 3) were added, acting during 4 h at room temperature. To reduce the lipofuscin-induced autofluorescence, the wholemounts were incubated in an ammonium acetate buffer (pH 5.0) containing 1 mM CuSO4 for 60–90 min followed by a brief dip in distilled H2O. Afterwards, the wholemounts were mounted in TBS-glycerol (1:1, pH 8.6). Wholemounts incubated in solutions without the primary antibodies (negative controls) were conducted to control the reaction. 2.3. Acquisition of images of the ganglia For the acquisition of ganglia images of the enteric nervous system, the nervous ganglia were randomly selected. Using confocal microscopy laser scanning (Bio-Rad MRC 1000 with a Nikon diaphot 300, equipped with a Argon Krypton laser, American Laser Corporation, Salt Lake City, UT), Z-series were created through the application of three wavelengths for the detection of secondary antibodies (488, 568, 647 nm of excitation; z-steps 0.6 lm). The 20 objective lens (numerical aperture 0.75) was used to locate the ganglia, while the 40 objective lens was used for the Z-series
Fig. 1. Peripherin structures by imunofluorescent test. Non-infected individual (A) presented preserved neuronal ganglia, with regular shaped neuronal bodies and no inflammation process signals. Chagasic patient (B) shown deformed ganglia and increased size of neuronal bodies (arrowhead).
Graphic 1. Morphometric analysis of Peripherin imuno-reactive neurons in colon samples from non-infected individuals group (gray) and chagasic patients with megacolon group (white). The values are expressed as means of immunoreactive areas ± SD. ⁄Statistically significant differences between this group and the non-infected individuals group. Total area of 1066 lm2 for all patients was analyzed (P 6 0.05).
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image acquisition, with the help of the Confocal Assistant 4.02 software. Images of the ganglia were prepared using Adobe Photoshop CS (8.0.1). 2.4. Statistical analysis Statistical analyses were conducted based on the nonparametric Anova-One way test, with the goal of detecting differences between the groups of patients. The significance level was p < 0.05 and all analyses were performed using the GraphPad Prim 3.0 software (San Diego, CA). The distribution of frequencies of all the variables and the measures of central tendency were calculated
using various parameters: average, mean, percentiles, standard deviation. The associations between the dependent and independent variables were tested through bivariate and multivariate regression techniques (simple linear regression, multiple or logistical regression, according to the characteristics of the variable). 3. Results 3.1. Innervation evaluation The innervation evaluation was performed by measurement of Peripherin immune-reactive (IR) structures. Qualitative and
Fig. 2. Characterization of the regenerative process of Calretinin, cChaT and Substance P in colon samples from chagasic patients with megacolon and non-infected individuals. Neuropeptides were marked by blue while the regeneration marker, GAP-43, was labeled by red. IPANs regeneration process (Calretinin + GAP-43) is demonstrated in non-infected individual (A) and chagasic patient with megacolon (B). Chagasic patients presented a small reduction in the regeneration process compared with non-infected individuals. The regeneration process in motor excitatory neurons was evaluated by cChat and GAP-43 ((C) non-infected individual and (D) chagasic patient with megacolon) and Substance P and GAP-43 ((E) non-infected individual and (F) chagasic patient with megacolon) combinations. Some neuronal filaments located in the muscle layer were demonstrated playing a regeneration process ((E and F) arrowheads), but these were not quantified for lack of an SP marking. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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quantitative analysis was executed in non-infected individuals and patients with Chagas’ disease. Non infected individuals presented preserved neuronal ganglia, with regular shaped neuronal bodies and no signal of inflammatory process, while chagasic patients shown deformed ganglia and increased neuronal bodies (Fig. 1). The statistical analyses exhibited a significantly reduced quantity of neurons and neuronal filaments Peripherin-IR when compared to the non-infected group (Graphic 1).
3.2. Analysis of the regenerative capacity of different neuronal classes The regeneration capacity of IPANs (Intrinsic Primary Afferent Neurons) was evaluated by combination of Calretinin and GAP-43 (Fig. 2). The results showed a small reduction of IPANs area
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in the regeneration process of patients with megacolon group compared to non-infected individuals (Graphic 2). The analysis of cChat and GAP-43 and Substance P and GAP-43 combinations showed a reduction in the regeneration process of excitatory motor neurons in the myenteric plexus from patients with megacolon when compared to the healthy individuals (Fig. 2), but this difference was not statistically significant (Graphic 3). Many neuronal filaments were found undergoing regeneration process in the muscular layer, but these were not quantified for lack of an Substance P IR marking. The samples marked with the association of NPY and GAP-43, to evaluate the regeneration process in the interneurons (Fig. 3, A and B) proved to be similar in chagasic patients and the healthy individuals (Graphic 2). Samples marked with VIP, NOS and GAP-43 combinations (Fig. 3), showed a significant increase in the rate of regeneration
Graphic 2. Morphometric analysis of Calretinin + GAP-43 and NPY + GAP-43 combinations. The values are expressed as means of immunoreactive areas ± SD. The analysis did not presented any statistically significant differences between the groups. Total area of 1066 lm2 for all patients was analyzed.
Graphic 3. Morphometric analysis of cChaT + GAP-43 and Substance P + GAP-43 combinations to evaluate the regeneration process in excitatory motor neurons. The values are expressed as means of immunoreactive areas ± SD. The analysis did not present any statistically significant differences between the groups. Total area of 1066 lm2 for all patients was analyzed.
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process of inhibitory motor neurons in chagasic patients with megacolon group when compared to non-infected individuals group (Graphic 4). 4. Discussion Megaesophagus and megacolon are the most common alterations in the digestive tract caused by Chagas’ disease [27]. Currently, denervation is accepted as one of the main megacolon development causes [17,28]. The neuronal destruction of Chagas’ disease in the acute phase occurs due to parasite concentration in the tissue, but in the chronic phase it is also related to the inflammatory process observed in this phase [25,29]. However,
the destruction process and the enteric nervous system response to this inflammatory process are still obscure. Thus, in order to clarify the processes surrounding the destruction of the ENS in patients with Chagas’ disease, the regeneration of different neuronal subclasses regeneration were evaluated in patients with megacolon. In this study, the innervated area was measured through the use of the Peripherin identification, a panneuronal marker, and our results showed a significant neuronal area reduction in samples for patients with chagasic megacolon. These data are in accord to previous studies on megacolon [17,22,25]. Previously, our group demonstrated that IPANs, excitatory motor neurons and interneurons were present in similar quantities for
Fig. 3. Characterization of the regenerative process of NPY, VIP and NOS in colon samples from chagasic patients with megacolon and non-infected individuals. Neuropeptides were marked by blue while the regeneration marker, GAP-43, was labeled by red. Interneurons regeneration process (NPY + GAP-43) is demonstrated in noninfected individual (A) and chagasic patient with megacolon (B). The regeneration process in inhibitory motor neurons was evaluated by VIP + GAP-43 ((C) non-infected individual and (D) chagasic patient with megacolon) and NOS + GAP-43 ((E) non-infected individual and (F) chagasic patient with megacolon) combinations. We observe that the regeneration process in both inhibitory motor neurons markers (VIP and NOS) are augmented in chagasic patients with megacolon ((D and F)) compared with noninfected individuals ((C and E)). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Graphic 4. Morphometric analysis of VIP + GAP-43 and NOS + GAP-43 combinations to evaluate inhibitory motor neurons. The values are expressed as means of immunoreactive areas ± SD. ⁄Statistically significant differences between this group and the non-infected individuals group. Total area of 1066 lm2 for all patients was analyzed (P 6 0.05).
healthy individuals and those with Chagas’ disease [17]. Here, the analysis of neuronal classes’ regenerative process of the neurons is similar in both chagasic patients and non-infected individuals, suggesting the destruction of these classes is not accentuated, and increase in the regenerative process is probably not necessary. Our research group has shown that there is an increased presence of excitatory motor neurons that synthesize substance P in patients with Chagas’ disease [17,30]. Substance P, aside from being a part of the neurotransmission process, also acts in the inflammatory response as a pro-inflammatory factor [31]. We believe the increase of SP in patients with chagasic megacolon may be an attempt by the organism to eliminate the parasite through an increase in the inflammatory response. In fact, a small reduction in the regenerative process of neurons that synthesize SP was observed in patients with megacolon. As these neurons are found in increased quantities in these patients, it was expected that the regenerative process would be reduced. Previous data from our lab suggested that the inhibitory motor neurons, marked with VIP and NOS were found in reduced quantities in Chagas’ disease patients [17]. The vasoactive intestinal peptide, aside from participating in the relaxing of the musculature of the colon, also has an anti-inflammatory role. The nitric oxide also participates in the relaxing of the colon, but in the inflammatory response it can act as an anti-inflammatory or a pro-inflammatory [32]. We believe that, because of the anti-inflammatory actions, the parasite would have greater freedom to attack these types of neurons. In the current study, we show that the regeneration process is increased in these neuronal classes, suggesting it might be a direct response of the host to compensate the inhibitory neurons loss caused by the parasite. Our results are in line with clinical observations that indicated a relaxing capacity loss in the large intestine from chagasic patients with megacolon. This loss produces colon motility changes, leading to feces accumulation and, consequently, its dilation. Besides, we can elude the incoordination of the rectum-sigmoid segment, hyperactivity of the cholinergic stimuli and achalasia of the internal anal sphincter [33]. Given these findings, we believe that in the regenerative process of inhibitory motor neurons (VIP and NOS) lies the key to the development of the chagasic megacolon. The complementation
of these results will come from further studies that seek the characterization of the effects and the cells that produce neurotrophins, which will allow us to know the exact conditions that permit the neuronal regenerative process to occur. Our results may enhance the perspective for the development of new drugs and therapeutic mechanisms to control the development of megacolon and even its prevention. Finally, we believe that our results can help the comprehension of Chagas’ disease, as well as other pathologies that affect the gastrointestinal tract. Acknowledgments This work was supported by funds from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), and Deutsche Forschungsgemeinschaft (BR 1815/4-1). This study does not represent a conflict of interests. References [1] Koberle F. Chagas’ disease and Chagas’ syndromes: the pathology of American trypanosomiasis. Adv Parasitol 1968;6:63–116. [2] Dias JC. Chagas disease, environment, participation, and the state. Cadernos de saude publica/Ministerio da Saude, Fundacao Oswaldo Cruz, Escola Nacional de Saude Publica 2001;17(Suppl.):165–9. [3] Romana C. The developmental cycle of Trypanosoma (Schizotrypanum) cruzi Chagas, in its tissular and hematic phases. Mem Inst Oswaldo Cruz 1909;1956(54):255–69. [4] Dias JC. General aspects of the prevention of Chagas’ disease in Brazil. Rev Paul Med 1984;102:279–81. [5] Matsuda NM, Miller SM, Szurszewski JH. Heme-oxygenase-2 immunolabelling in pig jejunum. Acta Histochem 2009. [6] Benchimol Barbosa PR. The oral transmission of Chagas’ disease: an acute form of infection responsible for regional outbreaks. Int J Cardiol 2006:112, 132–133. [7] Altcheh J. Chagas disease, 100 years after its identification. Arch Argent Pediatr 2010;108:4–5. [8] Campos JV, Tafuri WL. Chagas enteropathy. Gut 1973;14:910–9. [9] Reis DD, Jones EM, Tostes Jr S, Lopes ER, Gazzinelli G, Colley DG, et al. Characterization of inflammatory infiltrates in chronic chagasic myocardial lesions: presence of tumor necrosis factor-alpha+ cells and dominance of granzyme A+, CD8+ lymphocytes. Am J Trop Med Hyg 1993;48:637–44. [10] Corbett CE, Ribeiro Jr U, Prianti MG, Habr-Gama A, Okumura M, GamaRodrigues J. Cell-mediated immune response in megacolon from patients with chronic Chagas’ disease. Dis Colon Rectum 2001;44:993–8. [11] da Silveira AB, Adad SJ, Correa-Oliveira R, Furness JB, D’Avila Reis D. Morphometric study of eosinophils, mast cells, macrophages and fibrosis in
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journal homepage: www.elsevier.com/locate/humimm
Regenerative process evaluation of neuronal subclasses in chagasic patients with megacolon Milena Dionízio Moreira a, Axel Brehmer b, Enio Chaves de Oliveira c, Salustiano Gabriel Neto c, Alejandro O. Luquetti c, Lilian Lacerda Bueno d, Ricardo Toshio Fujiwara d, Michelle Aparecida Ribeiro de Freitas e, Alexandre Barcelos Morais da Silveira a,⇑ a
Neurosciences Laboratory, Human Anatomy Department, ICBIM, Universidade Federal de Uberlândia, Minas Gerais 38.400-902, Brazil Institute of Anatomy I, University of Erlangen–Nuremberg, Krankenhausstr. 9, Erlangen D-91054, Germany Department of Surgery, Medical School, Universidade Federal de Goiás, Goiânia, Goiás 74.605-020, Brazil d Department of Parasitology, ICB, Universidade Federal de Minas Gerais 31.270-901, Brazil e Parasitology Department, ICBIM, Universidade Federal de Uberlândia, Minas Gerais 38.400-902, Brazil b c
a r t i c l e
i n f o
Article history: Received 27 June 2012 Accepted 27 November 2012 Available online 5 December 2012
a b s t r a c t Chagas’ disease is one of the most serious parasitic diseases of Latin America, with a social and economic impact far outweighing the combined effects of other parasitic diseases such as malaria, leishmaniasis and schistosomiasis. In the chronic phase of this disease, the destruction of enteric nervous system (ENS) components leads to megacolon development. Previous data presented that the regeneration tax in the ENS neurons is augmented in chagasic patients. Although, there are several neuronal types with different functions in the intestine a detailed study about the regeneration of every neuronal type was never performed before. Therefore, the aim of this study was to evaluate the regeneration tax of every neuronal cell type in the ENS from chagasic patients with megacolon and non-infected individuals. A neuronal regeneration marker (GAP-43) was used in combination with a pan-neuronal marker (Peripherin) and several neuropeptides markers (cChat, Substance P, NPY, VIP and NOS), and it was considered as positive just with the combination of these markers. Our results demonstrated that the regeneration levels of cChat, Substance P, and NPY were similar in chagasic patients and non-infected individuals. However, levels of VIP and NOS neuropeptides were increased in chagasic patients when compared with noninfected individuals. We believe that the augment in the regeneration occur due to an increased destruction of selective neuronal types. These results corroborates with previous studies that pointed out to selective destruction of VIP and NOS neurons in chagasic patients. Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Chagas’ disease, caused by the Trypanosoma cruzi parasite, was discovered in 1909 by researcher Carlos Chagas, in Lassance, a city in the state of Minas Gerais, Brazil. It is estimated that currently 8 million people present the disease throughout the Americas, and is related to environmental and socio-political issues such as education, housing and migration [1,2]. In the last century, the main form of transmission of Chagas’ disease was by the hematophagous triatomine insect known as ‘‘kissing bug’’ [3]. In the last decades, aside from triatomine transmission, the parasite has also been transmitted via blood transfusions, organ transplants, via
⇑ Corresponding author. Address: Neurosciences Laboratory, Human Anatomy Sector, ICBIM, Campus Umuarama, Universidade Federal de Uberlândia, Minas Gerais 38400-902, Brazil. Fax: +55 34 3218 2472. E-mail address: [email protected] (A.B.M. da Silveira).
placenta and even via the consumption of foods contaminated by the parasite [4–6]. This disease presents two well defined phases: the acute phase and the chronic phase. The acute phase, lasting approximately two to three months, is characterized by an abundance of circulating parasites, fever, asthenia and malaise. After this phase, the individual goes into an asymptomatic state, which characterizes the beginning of the chronic phase. Years after the acute phase, the patient can continue not exhibiting symptoms, characterizing the indeterminate form of the disease, or can even develop pathological alterations in the functioning of the heart (cardiac form) and present gastrointestinal manifestations, culminating in the digestive form of the disease [1,3,7]. The digestive form of Chagas’ disease is manifested mainly as the megaesophagus and megacolon [1,8]. The megacolon is characterized by intestinal dilation associated with several focus of inflammatory process. Previous studies have shown that these focuses are made up of T CD3 lymphocytes, B CD20 lymphocytes and
0198-8859/$36.00 - see front matter Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humimm.2012.11.012
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natural killer cells and cytotoxic T lymphocytes. Nowadays, it is accepted that this inflammatory process is, at least in part, responsible for the destruction of components of the enteric nervous system [9–11]. The enteric nervous system is a division of the autonomous nervous system and represents the intrinsic innervation of the gastrointestinal tract. It is made up of neurons and cells incorporated in the intestinal wall. These neurons are located in the submucosa (submucosal plexus) and between the external and internal muscle layers (myenteric plexus), interconnected by bundles of neuronal filaments [12–15]. The enteric neurons can be identified by their function, morphology and neurochemical correlation. Functionally, they can be divided into: excitatory motor neurons, inhibitory motor neurons, interneurons and intrinsic primary afferent neurons (IPANs) [15]. The excitatory motor neurons innervate the longitudinal and circular smooth muscle of the entire digestive tract. The main transmitter of these neurons is acetylcholine (ACh) which acts in the muscle through muscarinic receptors. Tachykinins represented by the substance P (SP), contribute to the excitatory transmission, but have a secondary role with compared to that of ACh [15]. The inhibitory motor neurons release a combination of transmitters that contribute to the relaxation of the gastrointestinal tract. The primary neurotransmitter of these neurons is nitric oxide (NO) which receives the secondary contribution of other substances such as vasoactive intestinal peptide (VIP) and adenosine triphosphate (ATP). The interneurons form chains that run along the intestine and can be identified in all regions. This neuronal class has somastostatin and neuropeptide Y (NPY) as markers and, in some cases, serotonin (5-HT) [13,15]. The intrinsic primary afferent neurons (IPAN) are numerous, approximately 500 per millimeter and, by the immunohistochemical technique, its best marker is calretinin. They are directly sensitive to the mechanical and chemical stimuli of the intestinal mucosa and the sum of synaptic events caused by the transmission of IPAN result in the activation of numerous interneurons and motor neurons [13,16,17]. When these neurons suffer an aggression, they can present a process of regeneration known as neuronal plasticity. This process can be identified through a neuronal differentiation marker called growth associated protein-43 (GAP-43). GAP-43 is an integral membrane protein, existing in elevated levels when there is development or regeneration of neurons [18–21]. The characterization of SNE alterations in Chagas’ disease has been greatly emphasized in the last few years [11,17,19,22–26]. Previous results in our research group demonstrated that there is a significant rate of regeneration in patients that did not develop the megacolon. However, it remains to be seen whether this neuronal regeneration occurs in all neuronal subclasses or if it is restricted to some neuronal groups. Thus, the objective of this work is to characterize the intensity of neuronal regeneration in the subclasses of neurons of the SNE in patients with Chagas’ disease. We believe these results may contribute not only to partially elucidate the pathogenesis of Chagas’ disease, but also to the understanding of the physiological functioning of the gastrointestinal tract.
2. Material and methods 2.1. Patients Tissue samples from chagasic patients with megacolon and a non-infected group used in this study, were collected via surgery or necropsy at Medical School Hospital - Federal University of Goiás, by Dr. Enio Chaves Oliveira. Dilatation was diagnosed with plain abdominal X-ray, digital rectal examination, and barium enema. Previous consent was given by all individuals, parents or
legal guardians for inclusion in the research. The use of these samples for scientific purposes was previously authorized and approved by the Research Ethic Committee of the Universidade Federal de Uberlândia (No. 110/11). Patient data is summarized in Table 1. Samples were collected only from dilated segment, with at least 4 cm of length and variable tissue circumference according to the amount of dilatation. The samples were rinsed with phosphate buffered saline (pH 7.2–7.4) and further fixated. During fixation, the gut samples were distended with fixative solution (freshly diluted solution of 4% paraformaldehyde in phosphate buffered saline – pH 7.2–7.4) using a syringe. Then, the inflated segments were placed in fixative solution for 2–4 h. After this time, the inflated segment was opened, separated in small pieces and stored in 0.05% thimerosal in 0.1 M phosphate buffered saline (pH 7.2–7.4) at 4 °C. Alternately, short segments were pinned out in a Sylgard lined Petri dish and transferred to 4% formalin in 0.1 M phosphate buffer (pH 7.4) at room temperature for 2–3 h. After several washes in 0.05 M TRIS-buffered saline (TBS; pH 7.4), three longitudinal muscle/myenteric plexus wholemounts (2 cm length, 1 cm width) per segment (derived from each patient) were prepared. In the following day, the small segments of tissue were
Table 1 Patients data. Groups
Gender
Age
Megacolon
Diagnostic
Non-infected group
M
46
No
M
75
No
F
75
No
F
72
No
F
58
No
F
58
No
F
43
No
F
81
No
Rectal adenocarcinoma Sigmoid adenocarcinoma Sigmoid diverticular disease Sigmoid diverticular disease Sigmoid diverticular disease Sigmoid adenocarcinoma Sigmoid diverticular disease Sigmoid adenocarcinoma
F M M F M F F F M F F F F
57 62 60 62 58 76 43 69 59 61 53 48 58
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Chagasic patients with megacolon
Megacolon Megacolon Megacolon Megacolon Megacolon Megacolon Megacolon Megacolon Megacolon Megacolon Megacolon Megacolon Megacolon
Table 2 Primary antibodies. Antibody
Source
Code
Dilution
Anti-peripherin Anti-calretinin Anti-substanciap Anti-cChAT Anti-neuropeptideo Y Anti-NOS Anti-VIP Anti-Gap-43
INVITROGEN DAKO INVITROGEN Advanced targeting systems SIGMA SIGMA SIGMA SIGMA
A-21272 M7245 180091 AB-N34 N9528 N7280 V3508 G8043
1:1000 1:200 1:1000 1:500 1:200 1:100 1:500 1:500
M.D. Moreira et al. / Human Immunology 74 (2013) 181–188 Table 3 Secondary antibodies. Antibody
Company
Dilution (ll)
Donkey anti-goat ALEXA 488 Donkey anti-mouse ALEXA 555 Donkey anti-rabbit ALEXA 488
Molecular Probes Molecular Probes Molecular Probes
1:1000 1:1000 1:1000
transferred to a mixture of PBS-sucrose-azide and OCT compound (Tissue Tek, Elkhart, IN, USA) at a ratio of 1:1 for 24 h before being embedded in 100% OCT. Sections 12 lm thick were cut and mounted on microscope slides and dried for 1 h at room temperature. 2.2. Immunohistochemistry The wholemounts were pre-incubated for 2 h in TBS 0.05 M (pH 7.4) with 1% bovine serum albumin (BSA), 0.5% Triton X-100, 0.05% thimerosal and 5% goat serum. After being rinsed in TBS for 10 min, they were incubated in a solution containing BSA, Triton X-100, thimerosal and the primary antibodies (Table 2) for 24 h at 4 °C.
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In the next day, the wholemounts were washed in TBS and then the secondary antibodies (Table 3) were added, acting during 4 h at room temperature. To reduce the lipofuscin-induced autofluorescence, the wholemounts were incubated in an ammonium acetate buffer (pH 5.0) containing 1 mM CuSO4 for 60–90 min followed by a brief dip in distilled H2O. Afterwards, the wholemounts were mounted in TBS-glycerol (1:1, pH 8.6). Wholemounts incubated in solutions without the primary antibodies (negative controls) were conducted to control the reaction. 2.3. Acquisition of images of the ganglia For the acquisition of ganglia images of the enteric nervous system, the nervous ganglia were randomly selected. Using confocal microscopy laser scanning (Bio-Rad MRC 1000 with a Nikon diaphot 300, equipped with a Argon Krypton laser, American Laser Corporation, Salt Lake City, UT), Z-series were created through the application of three wavelengths for the detection of secondary antibodies (488, 568, 647 nm of excitation; z-steps 0.6 lm). The 20 objective lens (numerical aperture 0.75) was used to locate the ganglia, while the 40 objective lens was used for the Z-series
Fig. 1. Peripherin structures by imunofluorescent test. Non-infected individual (A) presented preserved neuronal ganglia, with regular shaped neuronal bodies and no inflammation process signals. Chagasic patient (B) shown deformed ganglia and increased size of neuronal bodies (arrowhead).
Graphic 1. Morphometric analysis of Peripherin imuno-reactive neurons in colon samples from non-infected individuals group (gray) and chagasic patients with megacolon group (white). The values are expressed as means of immunoreactive areas ± SD. ⁄Statistically significant differences between this group and the non-infected individuals group. Total area of 1066 lm2 for all patients was analyzed (P 6 0.05).
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image acquisition, with the help of the Confocal Assistant 4.02 software. Images of the ganglia were prepared using Adobe Photoshop CS (8.0.1). 2.4. Statistical analysis Statistical analyses were conducted based on the nonparametric Anova-One way test, with the goal of detecting differences between the groups of patients. The significance level was p < 0.05 and all analyses were performed using the GraphPad Prim 3.0 software (San Diego, CA). The distribution of frequencies of all the variables and the measures of central tendency were calculated
using various parameters: average, mean, percentiles, standard deviation. The associations between the dependent and independent variables were tested through bivariate and multivariate regression techniques (simple linear regression, multiple or logistical regression, according to the characteristics of the variable). 3. Results 3.1. Innervation evaluation The innervation evaluation was performed by measurement of Peripherin immune-reactive (IR) structures. Qualitative and
Fig. 2. Characterization of the regenerative process of Calretinin, cChaT and Substance P in colon samples from chagasic patients with megacolon and non-infected individuals. Neuropeptides were marked by blue while the regeneration marker, GAP-43, was labeled by red. IPANs regeneration process (Calretinin + GAP-43) is demonstrated in non-infected individual (A) and chagasic patient with megacolon (B). Chagasic patients presented a small reduction in the regeneration process compared with non-infected individuals. The regeneration process in motor excitatory neurons was evaluated by cChat and GAP-43 ((C) non-infected individual and (D) chagasic patient with megacolon) and Substance P and GAP-43 ((E) non-infected individual and (F) chagasic patient with megacolon) combinations. Some neuronal filaments located in the muscle layer were demonstrated playing a regeneration process ((E and F) arrowheads), but these were not quantified for lack of an SP marking. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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quantitative analysis was executed in non-infected individuals and patients with Chagas’ disease. Non infected individuals presented preserved neuronal ganglia, with regular shaped neuronal bodies and no signal of inflammatory process, while chagasic patients shown deformed ganglia and increased neuronal bodies (Fig. 1). The statistical analyses exhibited a significantly reduced quantity of neurons and neuronal filaments Peripherin-IR when compared to the non-infected group (Graphic 1).
3.2. Analysis of the regenerative capacity of different neuronal classes The regeneration capacity of IPANs (Intrinsic Primary Afferent Neurons) was evaluated by combination of Calretinin and GAP-43 (Fig. 2). The results showed a small reduction of IPANs area
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in the regeneration process of patients with megacolon group compared to non-infected individuals (Graphic 2). The analysis of cChat and GAP-43 and Substance P and GAP-43 combinations showed a reduction in the regeneration process of excitatory motor neurons in the myenteric plexus from patients with megacolon when compared to the healthy individuals (Fig. 2), but this difference was not statistically significant (Graphic 3). Many neuronal filaments were found undergoing regeneration process in the muscular layer, but these were not quantified for lack of an Substance P IR marking. The samples marked with the association of NPY and GAP-43, to evaluate the regeneration process in the interneurons (Fig. 3, A and B) proved to be similar in chagasic patients and the healthy individuals (Graphic 2). Samples marked with VIP, NOS and GAP-43 combinations (Fig. 3), showed a significant increase in the rate of regeneration
Graphic 2. Morphometric analysis of Calretinin + GAP-43 and NPY + GAP-43 combinations. The values are expressed as means of immunoreactive areas ± SD. The analysis did not presented any statistically significant differences between the groups. Total area of 1066 lm2 for all patients was analyzed.
Graphic 3. Morphometric analysis of cChaT + GAP-43 and Substance P + GAP-43 combinations to evaluate the regeneration process in excitatory motor neurons. The values are expressed as means of immunoreactive areas ± SD. The analysis did not present any statistically significant differences between the groups. Total area of 1066 lm2 for all patients was analyzed.
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process of inhibitory motor neurons in chagasic patients with megacolon group when compared to non-infected individuals group (Graphic 4). 4. Discussion Megaesophagus and megacolon are the most common alterations in the digestive tract caused by Chagas’ disease [27]. Currently, denervation is accepted as one of the main megacolon development causes [17,28]. The neuronal destruction of Chagas’ disease in the acute phase occurs due to parasite concentration in the tissue, but in the chronic phase it is also related to the inflammatory process observed in this phase [25,29]. However,
the destruction process and the enteric nervous system response to this inflammatory process are still obscure. Thus, in order to clarify the processes surrounding the destruction of the ENS in patients with Chagas’ disease, the regeneration of different neuronal subclasses regeneration were evaluated in patients with megacolon. In this study, the innervated area was measured through the use of the Peripherin identification, a panneuronal marker, and our results showed a significant neuronal area reduction in samples for patients with chagasic megacolon. These data are in accord to previous studies on megacolon [17,22,25]. Previously, our group demonstrated that IPANs, excitatory motor neurons and interneurons were present in similar quantities for
Fig. 3. Characterization of the regenerative process of NPY, VIP and NOS in colon samples from chagasic patients with megacolon and non-infected individuals. Neuropeptides were marked by blue while the regeneration marker, GAP-43, was labeled by red. Interneurons regeneration process (NPY + GAP-43) is demonstrated in noninfected individual (A) and chagasic patient with megacolon (B). The regeneration process in inhibitory motor neurons was evaluated by VIP + GAP-43 ((C) non-infected individual and (D) chagasic patient with megacolon) and NOS + GAP-43 ((E) non-infected individual and (F) chagasic patient with megacolon) combinations. We observe that the regeneration process in both inhibitory motor neurons markers (VIP and NOS) are augmented in chagasic patients with megacolon ((D and F)) compared with noninfected individuals ((C and E)). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Graphic 4. Morphometric analysis of VIP + GAP-43 and NOS + GAP-43 combinations to evaluate inhibitory motor neurons. The values are expressed as means of immunoreactive areas ± SD. ⁄Statistically significant differences between this group and the non-infected individuals group. Total area of 1066 lm2 for all patients was analyzed (P 6 0.05).
healthy individuals and those with Chagas’ disease [17]. Here, the analysis of neuronal classes’ regenerative process of the neurons is similar in both chagasic patients and non-infected individuals, suggesting the destruction of these classes is not accentuated, and increase in the regenerative process is probably not necessary. Our research group has shown that there is an increased presence of excitatory motor neurons that synthesize substance P in patients with Chagas’ disease [17,30]. Substance P, aside from being a part of the neurotransmission process, also acts in the inflammatory response as a pro-inflammatory factor [31]. We believe the increase of SP in patients with chagasic megacolon may be an attempt by the organism to eliminate the parasite through an increase in the inflammatory response. In fact, a small reduction in the regenerative process of neurons that synthesize SP was observed in patients with megacolon. As these neurons are found in increased quantities in these patients, it was expected that the regenerative process would be reduced. Previous data from our lab suggested that the inhibitory motor neurons, marked with VIP and NOS were found in reduced quantities in Chagas’ disease patients [17]. The vasoactive intestinal peptide, aside from participating in the relaxing of the musculature of the colon, also has an anti-inflammatory role. The nitric oxide also participates in the relaxing of the colon, but in the inflammatory response it can act as an anti-inflammatory or a pro-inflammatory [32]. We believe that, because of the anti-inflammatory actions, the parasite would have greater freedom to attack these types of neurons. In the current study, we show that the regeneration process is increased in these neuronal classes, suggesting it might be a direct response of the host to compensate the inhibitory neurons loss caused by the parasite. Our results are in line with clinical observations that indicated a relaxing capacity loss in the large intestine from chagasic patients with megacolon. This loss produces colon motility changes, leading to feces accumulation and, consequently, its dilation. Besides, we can elude the incoordination of the rectum-sigmoid segment, hyperactivity of the cholinergic stimuli and achalasia of the internal anal sphincter [33]. Given these findings, we believe that in the regenerative process of inhibitory motor neurons (VIP and NOS) lies the key to the development of the chagasic megacolon. The complementation
of these results will come from further studies that seek the characterization of the effects and the cells that produce neurotrophins, which will allow us to know the exact conditions that permit the neuronal regenerative process to occur. Our results may enhance the perspective for the development of new drugs and therapeutic mechanisms to control the development of megacolon and even its prevention. Finally, we believe that our results can help the comprehension of Chagas’ disease, as well as other pathologies that affect the gastrointestinal tract. Acknowledgments This work was supported by funds from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), and Deutsche Forschungsgemeinschaft (BR 1815/4-1). This study does not represent a conflict of interests. References [1] Koberle F. Chagas’ disease and Chagas’ syndromes: the pathology of American trypanosomiasis. Adv Parasitol 1968;6:63–116. [2] Dias JC. Chagas disease, environment, participation, and the state. Cadernos de saude publica/Ministerio da Saude, Fundacao Oswaldo Cruz, Escola Nacional de Saude Publica 2001;17(Suppl.):165–9. [3] Romana C. The developmental cycle of Trypanosoma (Schizotrypanum) cruzi Chagas, in its tissular and hematic phases. Mem Inst Oswaldo Cruz 1909;1956(54):255–69. [4] Dias JC. General aspects of the prevention of Chagas’ disease in Brazil. Rev Paul Med 1984;102:279–81. [5] Matsuda NM, Miller SM, Szurszewski JH. Heme-oxygenase-2 immunolabelling in pig jejunum. Acta Histochem 2009. [6] Benchimol Barbosa PR. The oral transmission of Chagas’ disease: an acute form of infection responsible for regional outbreaks. Int J Cardiol 2006:112, 132–133. [7] Altcheh J. Chagas disease, 100 years after its identification. Arch Argent Pediatr 2010;108:4–5. [8] Campos JV, Tafuri WL. Chagas enteropathy. Gut 1973;14:910–9. [9] Reis DD, Jones EM, Tostes Jr S, Lopes ER, Gazzinelli G, Colley DG, et al. Characterization of inflammatory infiltrates in chronic chagasic myocardial lesions: presence of tumor necrosis factor-alpha+ cells and dominance of granzyme A+, CD8+ lymphocytes. Am J Trop Med Hyg 1993;48:637–44. [10] Corbett CE, Ribeiro Jr U, Prianti MG, Habr-Gama A, Okumura M, GamaRodrigues J. Cell-mediated immune response in megacolon from patients with chronic Chagas’ disease. Dis Colon Rectum 2001;44:993–8. [11] da Silveira AB, Adad SJ, Correa-Oliveira R, Furness JB, D’Avila Reis D. Morphometric study of eosinophils, mast cells, macrophages and fibrosis in
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