INTRODUCTION
Schistosomiasis mansoni is one of the most prevalent neglected tropical water-borne diseases in populations living in low socio-economic and sanitary conditions (Gryseels et al. Reference Gryseels, Polman, Clerinx and Kestens2006). It is estimated that about 207 million people are infected worldwide, in which 120 million are symptomatic and 20 million have serious consequences (Chitsulo et al. Reference Chitsulo, Engels, Montresor and Savioli2000). Morbidity observed in schistosomiasis is linked to the intense granulomatous reaction to trapped eggs in host organs such as liver, intestine, lungs, brain and pancreas (Baptista and Andrade, Reference Baptista and Andrade2005). Acute schistosomiasis is a clinical syndrome often seen in non-immune individuals 3 to 6 weeks after exposure during a bath in an endemic area (Epelboin et al. Reference Epelboin, Jauréguiberry, Estève, Danis, Komajda, Bricaire and Caumes2010). With ageing of infection, some patients may progress to develop the severe hepatosplenic form of the disease. Clinical presentation includes hepatosplenomegaly, periportal fibrosis, portal hypertension, ascites, oesophageal varices, collateral circulation and haematemesis (Gryseels et al. Reference Gryseels, Polman, Clerinx and Kestens2006; Lambertucci, Reference Lambertucci2010).
Even though cardiac involvement during acute schistosomiasis is uncommon, myocarditis associated with or without granuloma and right ventricular insufficiency has been reported (Chisty et al. Reference Chisty, Nargis, Sato, Inaba and Kamiya1999; Ramanampamonjy et al. Reference Ramanampamonjy, Razafimahefa, Rajaonarivelo and Rajaona2007). Recently, electrocardiography showed myocarditis as a complication of acute schistosomiasis in non-immune travellers returning from endemic areas (Epelboin et al. Reference Epelboin, Jauréguiberry, Estève, Danis, Komajda, Bricaire and Caumes2010). Other image examinations showed that schistosomiasis-associated pulmonary arterial hypertension (PAH) also shows cardiac alterations, as right-side cardiomegaly, leading to a heart failure (Kolosionek et al. Reference Kolosionek, Graham, Tuder and Butrous2011).
Since the mid-1990s, a number of studies have used unbiased stereological methods to quantify heart morphology (Wulfsohn et al. Reference Wulfsohn, Nyengaard and Tang2004). Design-based stereology can provide fundamental information on the structural changes in the pathological heart (Medeiros et al. Reference Medeiros, Mothé, Águila and Mandarim-De-Lacerda2005). There is growing evidence that a high-fat chow contributes to increased myocardial injury. Previous work in rats fed lard and egg yolk showed cardiomyocyte hypertrophy, microcirculation rarefaction and myocardial ischaemia with consequent cardiomyocyte loss and interstitial fibrosis (Águila and Mandarim-de-Lacerda, Reference Águila and Mandarim-De-Lacerda2001).
Considerable advances have been made in recent years in our knowledge on the role of dyslipidaemia associated with both acute and chronic liver and intestinal pathology in schistosomiasis by utilizing the stereological approach. These studies have shown lower hepatocyte and sinusoid numbers and higher liver steatosis (Neves et al. Reference Neves, Alencar, Águila, Mandarim-De-Lacerda, Machado-Silva and Gomes2006), as well as a higher granulomatous reaction in the small intestine compared to mice fed a normal chow (Alencar et al. Reference Alencar, Neves, Águila, Mandarim-De-Lacerda, Gomes and Machado-Silva2009). Morphometric studies also showed that the infected high-fat chow group had significant alterations in small intestine structure (De Barros Alencar et al. 2012). Much remains to be learned, however, about the burden of cardiac involvement in S. mansoni-infected mice fed a high fat chow. This study aimed to investigate myocardial injuries in these double burdens.
MATERIALS AND METHODS
Experimental protocol
The study design reported herein complies with the current laws regarding ethical procedures involving laboratory animals and was approved by Comissão de Ética de Uso de Animais-Fiocruz (L-0036/07), Rio de Janeiro, Brazil.
The animals, chow regimen, infection and biochemical analysis have been previously reported (Alencar et al. Reference Alencar, Neves, Águila, Mandarim-De-Lacerda, Gomes and Machado-Silva2009). Briefly, female Swiss Webster mice were fed either a high-fat chow or a control chow for 6 months. Later, the mice were given 50 S. mansoni cercariae transcutaneously. Total serum cholesterol was assayed 1 day before experimental infection and 63 days post-infection. According to the chow regimen and infection (acute or chronic), mice were separated into 4 groups (n = 5 each/group) as follows: uninfected mice fed standard chow, uninfected mice fed high-fat chow, infected mice fed standard chow and infected mice fed high-fat chow.
Mice were euthanized on the 9th week (acute phase) and 17th week (chronic phase) post-infection to remove organs such as the heart. The heart was weighed and its volume determined by Scherle´s liquid displacement method (Scherle, Reference Scherle1970).
Histological procedures
Hearts were fixed in modified Millonig's phosphate-buffered formalin, pH 7·4 for 48 h at room temperature prior to tissue procedures. Hearts were sectioned by the modified orthotopic method, separating the organ's middle region (Mandarim-de-Lacerda, Reference Mandarim-De-Lacerda2003). Those fragments were embedded in paraffin and sectioned to 6μm thickness before staining with Haematoxylin-Eosin (vessels) or Picrosirius red (myocytes) (Rich and Whittaker, Reference Rich and Whittaker2005) for general morphology, morphometry and stereology.
Histopathology and quantitative morphology
The Haematoxylin-Eosin (H&E) stained-section from intact intramyocardial vessels found in the middle region heart cutting was further investigated. We used the Image Pro Plus – Media Cybernetics (USA) for measurement of total myocyte area (μm2), average lumen diameter (μm) and mean wall thickness (left, right and bottom) (μm). Only cardiomyocytes (15–30 per animal) sectioned transversely, from the right ventricle and showing rounded shape were considered. Thus, we obtain a more uniform set of cardiomyocytes in all groups. The average cross-sectional areas obtained for each group were used as an indicator of cell size (Zornoff et al. Reference Zornoff, Matsubara, Matsubara, Minicucci, Azevedo, Camapanha and Paiva2006).
Picrosirius red-stained heart sections were observed under a polarized light microscope (Olympus BX51). Photomicrographs were analysed in Image Pro Plus software to quantify the percentage of non-fibril and fibril collagen in this tissue. The collagen percentage per area with dimensions 1360 × 1024 pixels was determined.
Stereological study
The stereological study applied was the optical disector (Sterio, Reference Sterio1984). It was used to estimate the numerical density of cardiomyocytes (Nv[c]), determined by Nv[c] = Q−/t.AT 1/mm3, where Q− is the number of cardiomyocyte nuclei seen in focus only in the look-up plane, t the thickness of the disector and AT the test area. The total number of cardiomyocyte nuclei per heart (N[c]) was estimated by the product of the numerical density of the cardiomyocyte nuclei by the previously measured volume of the heart (determined by Scherle's method) (Mandarim-de-Lacerda, Reference Mandarim-De-Lacerda2003; Fernandes-Santos et al. Reference Fernandes-Santos, de Souza Mendonça and Mandarim-de-Lacerda2009).
Statistical analysis
All summary data for continuous variables were expressed as mean±s.d. All analyses were performed with Instat software, in which groups were compared using variance analysis ANOVA. Values of P ⩽ 0·05 were regarded as statistically significant (Zar, Reference Zar1999).
RESULTS
Histopathology study
Histopathological examination of routine H&E cardiac sections revealed that infected control chow fed mice from the acute phase, compared to uninfected exposed controls (Fig. 1A and C), showed myocarditis characterized by some inflammatory foci, disappearance of fibres and fibroblast proliferation (Fig. 2A and B). Animals from the chronic phase developed more severe inflammation and regenerative changes than those in the acute phase (Fig. 3A).
Photomicrographs of the heart tissue in uninfected mice fed standard chow (A and C) and high-fat chow (B and D), stained with Haematoxylin-Eosin (A and B) and Picrosirius red (C and D). (A) Normal cardiac tissue of standard chow group (SC) with an intact blood-vessel. (B) High-fat group (HFC) with cardiac fibre loss and fibroblast proliferation (arrow). (C) Normal blood vessel with a thin collagen layer around of standard chow group. (D) Blood-vessel with a lot of collagen around of high-fat chow group. Scale bar = 15 μm.
Photomicrographs of the heart tissue in Schistosoma mansoni-infected mice fed standard chow (A and B) and high-fat chow (C-F) euthanized at acute phase, stained with Haematoxylin-Eosin. (A) Inflammatory focus (arrow) in standard chow group (ISCa). (B) Fibre loss and fibroblast alignment (arrows) in ISCa group. (C, D, E and F) High-fat chow group (IHFCa) with fat cell infiltration (+) (C); inflammatory focus (arrow) (D); coagulation of cardiac fibres (arrows) (E) and cardiac fibre loss and fibroblast proliferation (F). Scale bar = 15 μm (A-E) and 6 μm (F).
Photomicrographs of the heart tissue in Schistosoma mansoni-infected mice fed standard chow (A) and high-fat chow (B-D) euthanized at chronic phase, stained with Haematoxylin-Eosin (A-C) and Picrosirius red (D). (A) Standard chow group (ISCc) with inflammatory cells, fibre loss and fibroblast proliferation. (B, C and D) High-fat chow group (IHFCc) with large inflammatory foci (B); more fat cells infiltrating the cardiac tissue (*) than in IHFCa (Fig. 2-C) (C); and presence of an egg of S. mansoni (D). Scale bar = 15 μm (A-C) and 60 μm (D).
High-fat chow-fed mice showed cardiac fibre loss and fibroblast proliferation to perform local regeneration, but no inflammatory foci were detected. In addition, marked collagen deposition was observed surrounding intramyocardial vessels thicker than those analysed in uninfected standard chow-fed mice (Fig. 1B and D). All infected mice with high blood cholesterol from acute and chronic infection showed myocarditis characterized by a greater amount of inflammatory foci compared to standard chow-fed mice. Several areas displaying cardiomyocyte coagulation, extensive areas with fibre loss and fibroblast proliferation near blood vessels were identified in high-fat chow-fed mice. This group also presented inflammatory cells permeating the fibres (Fig. 2D-F and Fig. 3B). These animals contained a greater number of adipocytes deposited in tissue compared to their controls (Fig. 2C and Fig. 3C). A chronically high-fat infected mouse had an S. mansoni egg in its heart tissue, with extensive collagen deposition (Fig. 3D).
Quantitative studies
Intramyocardial vessels
High-fat fed infected mice from the acute phase (Table 1) displayed a narrower lumen diameter (P = 0·1895) than those fed the standard chow. The medial vessel wall thickness was significantly thicker (P = 0·0046) in infected mice than in controls, although in high-fat-fed infected mice it was 10·5% thicker than its control.
Quantitative data (mean±s.d.) of acute schistosomiasis (euthanasia after 42 weeks of experiments and 9 weeks post-infection) and its controls in standard or high-fat chow-fed mice
In the chronic phase of schistosomiasis (Table 2), lumen diameter (P = 0·8455) and vessel wall thickness (P = 0·0573) were not significantly different between infected groups and their controls, but the measurements were lower than those reported for the acute phase (Table 1). Even so, the blood vessel walls from infected animals were thicker than those of uninfected mice.
Quantitative data (mean±s.d.) of chronic schistosomiasis (euthanasia after 50 weeks of experiment and 17 weeks post-infection) and its controls in standard or high-fat chow-fed mice
Cardiomyocytes
All high-fat chow-fed mice showed a significantly (P < 0·0001) larger area than those fed a standard chow (Tables 1 and 2). In the acute phase (Table 1), the cardiomyocyte area from infected standard chow-fed mice was larger (+21·9%; P < 0·001) than its control. Comparing infected high-fat chow-fed mice and their control, no significant difference was found. Uninfected high-fat chow-fed mice showed an area of cardiac cells that was significantly (+40·7%; P < 0·001) increased compared to that of those fed a standard chow diet. Infected high-fat chow-fed mice showed an area of cardiac cells that was significantly (+25·9%; P < 0·001) increased compared to that of those fed a standard chow diet.
In the chronic phase (Table 2), it was observed that high-fat mice showed higher hyperplasia (+32%; P < 0·001) than those fed on standard chow. Infected high-fat chow-fed mice showed a larger increased area (+29%; P < 0·001) than the control.
Collagen
In the acute infection, infected standard chow-fed mice (+53%; P < 0·05) and high-fat chow-fed mice (+38%; P < 0·01) presented a higher amount of collagen per area than their controls. Uninfected high-fat chow-fed mice presented more collagen per area (+51%; P < 0·05) than standard chow-fed mice, as well as infected high-fat chow-fed mice (+35%; P < 0·05) compared to those infected and fed a control chow diet (Table 1).
In the chronic phase (Table 2), the amount of collagen in the cardiac tissue of all groups increased compared to the acute phase (P = 0·0024). Infected standard chow-fed mice (+47%) and high-fat-fed mice (+2%) exhibited more collagen than the uninfected mice. Comparing the high-fat chow group to the control group, collagen was about 60% higher in uninfected and 25% higher in infected than in control chow-fed mice.
Stereological study
All standard chow-fed mice from the acute infection showed a higher numerical density of cardiomyocytes (Nv[c]; P < 0·0001) and a higher total number of cardiomyocytes (N[c]; P = 0·0104) compared to animals fed high-fat chow (Table 1). Infected standard chow mice presented Nv[c] reduced (−32·6%; P < 0·001), whereas N[c] was (−25·6%; P > 0·05) reduced compared to control. Results from infected mice submitted to the high-fat chow were lower {(Nv[c] −13·7%; P < 0·05) and (N[c] −2·8%; P > 0·05)} than the uninfected control. Comparing infected high-fat chow-fed mice with standard chow-fed mice, we observed lower Nv[c] (−28·6%; P < 0·001) and N[c] (−34·8%; P > 0·05) compared to those from the control chow-fed mice.
In the chronic phase (Table 2), similar results to the previous phase were observed. Animals fed high-fat chow presented lower values of Nv[c] (P = 0·0009) and N[c] (P = 0·0028) compared to standard chow. Infected standard chow-fed mice presented lower Nv[c] (−41·9%; P < 0·001) and N[c] (−41·3%; P < 0·001) than the control group. Infected mice fed high-fat chow also showed Nv[c] reduced (−21·3%; P > 0·05) and N[c] decrease (−13·8%; P > 0·05) compared to uninfected mice. Comparing results from both chows, uninfected standard chow-fed mice presented higher N[c] (+46%; P < 0·001) than the high-fat chow-fed group. Infected high-fat-fed mice presented reduced N[c] (−20·6%; P < 0·001) compared to those fed standard chow.
DISCUSSION
Chow-induced models have proven beneficial for the evaluation of physiological changes that take place during the pathogenesis of diseases (Neves et al. Reference Neves, Alencar, Águila, Mandarim-De-Lacerda, Machado-Silva and Gomes2006). Most cardiovascular research focuses on the knowledge of morphology and functionality of healthy versus sick hearts (Tang et al. Reference Tang, Nyengaard, Andersen, Baandrup and Gundersen2009). Among other factors, poor nutritional conditions (Thone-Reineke et al. Reference Thone-Reineke, Kalk, Dorn, Klaus, Simon, Pfab, Godes, Persson, Unger and Hocher2006) and fat-rich chows (Águila et al. 1998) are important causes of heart injury. This study was designed to answer some queries: do mice infected by Schistosoma mansoni display cardiac involvement? To answer that, histopathological, stereological and morphometric analyses were performed in acute and chronic murine schistosomiasis.
Our results clearly confirm cardiac involvement under experimental conditions. Even though S. mansoni adults and eggs are not commonly found in heart tissue, histopathological examination of cardiac sections revealed that infected mice fed control chow from both acute phase and chronic infection showed myocarditis. This was comprised of some inflammatory foci, disappearance of fibres, fibroblast proliferation and septal thickening of interstitial tissue among cardiomyocytes. Over the last several years, we have used design-based stereology that provides fundamental information on the structural changes in the pathological liver (Neves et al. Reference Neves, Alencar, Águila, Mandarim-De-Lacerda, Machado-Silva and Gomes2006; Barros et al. Reference Barros, Costa-Silva, Biolchini, Neves and Machado-Silva2009; Machado-Silva et al. Reference Machado-Silva, Neves, Mota and Mandarim-de-Lacerda2010).
In this study, both lower numerical density and total number of cardiomyocytes were reduced in mice fed high-fat chow, compared with those fed normal chow. Furthermore, morphometry analysis confirmed that parasitized mice underwent cellular hypertrophy, whereas the wall thickness of intramyocardial vessels was increased. Even though heart damage during acute schistosomiasis mansoni is uncommon, myocarditis associated with or without granuloma and right ventricular insufficiency has been reported (Chisty et al. Reference Chisty, Nargis, Sato, Inaba and Kamiya1999; Ramanampamonjy et al. Reference Ramanampamonjy, Razafimahefa, Rajaonarivelo and Rajaona2007). Recently, electrocardiography showed myocarditis as a complicating acute schistosomiasis in non-immune travellers returning from endemic areas (Epelboin et al. Reference Epelboin, Jauréguiberry, Estève, Danis, Komajda, Bricaire and Caumes2010). The mechanism to account for the myocarditis remains to be elicited.
In the present study, aged animals (around 12 months) had fewer cardiomyocytes than younger mice, suggesting that the ageing process facilitates cardiomyocyte apoptosis as seen by Águila et al. 1998. As the infection advances to chronicity, the host tries to delimit parasites or egg-induced cellular reactions (Borojevic, Reference Borojevic1992), in which host responsiveness is diminished (Boros, Reference Boros1989). Our study demonstrated that chronically-infected mice developed higher cardiac pathology than those in the acute phase. Previous studies showed that ageing of the infection leads to alterations in the hepatic vasculature due to the deposited eggs (Silva et al. Reference Silva, Ribeiro-Dos-Santos, Soares and Andrade2006), which is associated with changes in the immunomodulation of liver granulomas (Alencar et al. Reference Alencar, Neves, Águila, Mandarim-De-Lacerda, Gomes and Machado-Silva2009). In the current study, cardiomyocyte necrosis continues throughout the course of the disease, resulting in an accumulation of extracellular matrix (fibrosis). Previous studies have demonstrated human cases of endomyocardial fibrosis (Rashwan et al. Reference Rashwan, Ayman, Ashour, Hassanin and Zeina1995; Victor et al. Reference Victor, Lira, Arruda, Monteiro and Lima1996), a disorder characterized by collagen enhancement along the tissue and surrounding vessels.
Another issue that remains to be understood is the burden of cardiac involvement in S. mansoni-infected mice fed a high-fat chow diet. Feeding mice with a high saturated fat chow, as used in our study, increased cardiomyocyte apoptosis and adipocyte deposits within the heart tissue (Okere et al. Reference Okere, Chandler, McElfresh, Rennison, Sharov, Sabbah, Tserng, Hoit, Ernsberger, Young and Stanley2006). The myocardium of spontaneously hypertensive rats displays morphological changes indicating tissue overload, such as myocyte loss, whereas the remainder undergo hypertrophy, reduced vascularization and ischaemia (perimysial and interstitial fibrosis) (Medeiros et al. Reference Medeiros, Mothé, Águila and Mandarim-De-Lacerda2005; Fernandes-Santos et al. Reference Fernandes-Santos, de Souza Mendonça and Mandarim-de-Lacerda2009). Our results showed that high-fat chow caused cardiomyocyte loss, cellular hypertrophy, thickening of vessel walls, lumen diameter reduction and disruption of the cardiac tissue. Those data are consistent with the findings in rats (Águila et al. Reference Águila, Mandarim-De-Lacerda and Apfel1998; Águila and Mandarim-de-Lacerda, Reference Mandarim-De-Lacerda2003) and obese mice (Pinheiro et al. Reference Pinheiro, Cunha, Águila and Mandarim-De-Lacerda2007).
The heart injuries were worsened in all infected mice with high blood cholesterol from acute and chronic infection compared to mice fed standard chow. There was a greater amount of inflamed tissue, and reduction in the number and hypertrophy of cardiomyocytes. In addition, both necrosis events and cell death, resulting from fibre coagulation were increased. The increased number of fibroblasts that were permeating the coagulated and disappearing cardiomyocyte, led to increased collagen production throughout the tissue, characterizing ischaemic heart disease (Taube and Hutchins, Reference Taube and Hutchins2008). Our quantitative morphology showed that mice from acute infection presented a higher collagen content and wall thickness than uninfected mice and those fed control chow. Fibroblast proliferation and collagen synthesis are essential to maintain the myocardium form and structure (Palaniyandi et al. Reference Palaniyandi, Sun, Ferreira and Mochly-Rosen2009). Probably, excessive lipid accumulation in cardiomyocytes and infection may account for the cardiac disorder.
Though chronically-infected hosts have lower responsiveness than those from the acute infection, it may correspond to severe morbidity, given that enhanced collagen and fibrosis account for histopathological alterations (Boros, Reference Boros1989; Borojevic, Reference Borojevic1992). Furthermore, the long-term feeding of a high-fat chow has an effect on the outcome of chronic infection (Alencar et al. Reference Alencar, Neves, Águila, Mandarim-De-Lacerda, Gomes and Machado-Silva2009). Microscopic examination from the chronically-infected high-fat chow group revealed severe cardiac damage due to myocarditis, extensive collagen content and fibrosis, which occurs at the site of the granulomatous inflammatory response and enhances the disease pathology (Boros, Reference Boros1989). Quantification of numerical density and total number of cardiomyocytes showed significant reductions when comparing standard chow-fed mice euthanized at acute infection with those fed rich-lipid chow at chronic infection.
At this time, reparation of cardiac tissue is a strategy intended to promote the impairment in tissue structure, thereby improving associated functional derangements. Morphometry demonstrated that the collagen area was enhanced compared to control chow mice both at the acute and chronic infection phases. Otherwise, lumen diameter was reduced, while wall thickness was enhanced. Previous studies have demonstrated that adult worms significantly reduced atherogenesis in apolipoprotein E gene knockout mice (Doenhoff et al. Reference Doenhoff, Stanley, Griffiths and Jackson2002). The lumen vessel is bigger in infected, high-fat chow fed mice compared to uninfected mice. Recently, it was demonstrated that chronic exposure to schistosome eggs had no effect on atherosclerotic lesion development (La Flamme et al. Reference La Flamme, Harvie, Kenwright, Cameron, Rawlence, Low and McKenzie2007).
An egg was detected in the heart tissue. This notable feature provided by our data indicates that schistosome eggs can reach heart tissue without the associated hepatosplenomegaly and pulmonary hypertension (Warren, Reference Warren1964; Lapa et al. Reference Lapa, Dias, Jardim, Fernandes, Dourado, Figueiredo, Farias, Tsutsui, Terra-Filho, Humbert and Souza2009). These heart injuries may be associated with adult worms (Medeiros and Andrade, Reference Medeiros and Andrade1986) or toxic products from hepatic and intestinal granulomas (Rashwan et al. Reference Rashwan, Ayman, Ashour, Hassanin and Zeina1995; Carneiro et al. Reference Carneiro, Santos, Brant, Rabelo, Ligeiro, Barcelos, Silva, Silva and Nunes2011) that reach cardiac tissue due to collateral circulation. Data from the literature suggest that S. mansoni eggs can be retained in successive thrombotic formations that reach the heart causing endomyocardial fibrosis in human patients (Carneiro et al. Reference Carneiro, Santos, Brant, Rabelo, Ligeiro, Barcelos, Silva, Silva and Nunes2011). In conclusion, the collected data support the view that a double burden has a synergistic deleterious effect on myocardial tissue.
ACKNOWLEDGMENTS
The authors wish to thank Dr Lygia R. Corrêa from the Laboratory of Malacology, Fiocruz, for providing cercariae and Thatiany Marinho from the Laboratory of Morphometry, Metabolism & Cardiovascular Disease, Institute of Biology, UERJ, for histological sections.
FINANCIAL SUPPORT
Research supported by Fundação de Apoio à Pesquisa do Estado do Rio de Janeiro (Faperj, E-26/111.538/2010; E-26/101.965/2009) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes).
