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Tissue and Cell 40 (2008) 293–298

Morphological changes and EGF expression in the granular convolutedtubule cells of submandibular glands of Trypanosoma cruzi infected rats

A. Moreira a, M.H. Napimoga a, B.B. Benatti a, G.A.B. Silva b, D.B. Rocha-Rodrigues a,J.T. Clemente-Napimoga a, Gustavo A. Vieira a, J.B. Alves a,∗

a Laboratory of Molecular Biology, University of Uberaba, Av. Nene Sabino, 1801, Aeroporto, CEP 38.055-500 Uberaba, MG, Brazilb Institute of Biological Science, Federal University of Minas Gerais, Brazil

Received 7 November 2007; received in revised form 5 February 2008; accepted 20 February 2008Available online 10 April 2008

bstract

We have previously demonstrated in rats that Chagas’ disease affects the salivary glands, by promoting an enlargement of the submandibularland. In order to further investigate possible functional alterations on infected submandibular glands, the objective of the present study waso analyze epidermal growth factor (EGF) expression on rat submandibular glands during Trypanosoma cruzi infection. Results demonstratedhat infected rats presented lower levels of testosterone, and morphological changes in the granular convoluted tubule (GCT) cells of theubmandibular glands, along with acinar enlargement and delayed ductal maturation at the developing granular ducts. Immunohistochemistrynalysis additionally showed that only few cells immunolabelled with anti-EGF on infected rats during the acute phase of Chagas’ disease,hile after 64 and 90 days (chronic phase) of infection, EGF expression was similar to non-infected rats. The present findings suggest that at

he acute phase of Chagas’ disease, lower levels of testosterone may lead to a delayed maturation of GCT, which positively correlates withecreased EGF production by submandibular glands cells.

2008 Elsevier Ltd. All rights reserved.

eywords: Submandibular gland; Trypanosoma cruzi; Epidermal growth factor; Chagas’ disease

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. Introduction

Chagas’ disease is caused by the flagellate protozoonrypanosoma cruzi, which is transmitted by the feces oflood-sucking insect vectors (Triatoma). The parasite playsfundamental role by inducing immunopathology and tissueamage in organs such as the heart, esophagus, and colony sequentially inducing inflammatory responses, cellularesions, and fibrosis (Teixeira et al., 2002).

Besides the involvement of the cardiovascular and diges-

ive system, we have previously demonstrated that thisisease also affects the salivary glands, by promoting annlargement of the submandibular gland in rats (Alves and

Abbreviations: EGF, epidermal growth factor; NGF, nerve growth factor;CT, granular convoluted tubule.∗ Corresponding author. Tel.: +55 31 9984 70 76; fax: +55 34 3314 89 10.

E-mail address: propg.pr@uniube.br (J.B. Alves).

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040-8166/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.tice.2008.02.004

achado, 1980). This observation was more evident dur-ng the acute phase of experimental Chagas’ disease, inhich the absence of appreciable inflammatory process in

he submandibular glands of T. cruzi infected rats induced aevere reduction in sympathetic (Machado et al., 1984) andarasympathetic innervation (Alves and Machado, 1984). Athe histological level, the main changes observed were accel-rated acinar development and delayed ductal maturation,haracteristics that were more evident in the developing gran-lar ducts (Alves and Machado, 1980). On the other hand,uring the chronic phase of experimental Chagas’ diseasehe acini returned to its normal size and the granular ducthowed complete pattern recuperation (Alves et al., 1995).owever, the underlying mechanisms are still unknown.

It has been demonstrated that some factors produced by

ubmandibular glands may stimulate lymphocyte prolifera-ion, affect the weight of the thymus, spleen and lymph nodesnd induce immunosuppression in several in vivo animal

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odels (Sabbadini and Berczi, 1995). The submandibularlands produce significant quantities of nerve growth fac-or (NGF), epidermal growth factor (EGF), transformingrowth factor-beta and kallikreins, which are secreted intohe saliva and affect immune and mucosal tissues, and alsoerve endings in the gastrointestinal tract. These factors playmportant roles in regulating mucosal immuno/inflammatoryesponse and in regeneration and healing (Sabbadini anderczi, 1995).

Thus, the objective of the present investigation was toetermine by using morphological and immunohistochem-cal techniques the evolution of granular cell ducts of theubmandibular glands and the EGF expression during exper-mental Chagas’ disease in rats.

. Materials and methods

.1. Animal inoculation

Forty male Holtzman rats, aged 27–29 days, werenoculated intraperitoneally with 0.15 ml of mouse bloodontaining about 300,000 trypomastigotes of Y strain of T.ruzi. This strain, isolated from a patient with Chagas’ dis-ase (Silva and Nussenzweig, 1953), had been maintained inice by repeated blood passages every 7 days. The infectionas confirmed by the presence of living trypomastigotes in

he blood of all inoculated animals 10 days after inoculation.This animal study was deemed to be ethical according

o the Brazilian Guidelines (Resolution 196 of the Nationalealth Council, 1996), and the protocol was approved by theesearch Ethics Committee of Federal University of Minaserais—UFMG.

.2. Histological and immunohistochemical study

The animals were sacrificed under deep anesthesia after8, 32, 64 and 90 days of T. cruzi inoculation. The rightnd left submandibular glands were dissected, fixed in 10%uffered neutral formalin for 48 h, briefly washed in runningap water, dehydrated and embedded in paraffin wax. Eachample was sliced into 6 �m sections in sagittal directions.ections were mounted on glass slides and stained withematoxylin and eosin to analyze the tissue organization ofhe gland.

Additional sections were mounted on glass slides and usedor immunohistochemical analysis, immunolabelled for EGFSanta Cruz Biotechnology, Santa Cruz, CA, USA; 1:100).he slides were pre-treated with 3-aminopropyltriethoxy-ilane (Sigma, St. Louis, MO, USA) immersed 3 min inylene to eliminate the paraffin, dehydrated in absolute alco-ol, and re-hydrated with Tris-buffered saline (TBS). The

ections were rinsed in TBS and immersed in 3% hydrogeneroxide in methanol for 10 min to block the endogenouseroxidase activity and incubated for 30 min at 90 ◦C for anti-en recover. The slides were then incubated with monoclonal

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ell 40 (2008) 293–298

GF antibody for 2 h at 37 ◦C and after rinsed with TBS threeimes for 3 min.

The secondary biotinylated antibody (Vector Laborato-ies, Burlingame, CA, USA) was then applied on the sections,hich were incubated for 30 min and rinsed with TBS three

imes for 3 min. The streptavidin–biotin–peroxidase com-lex (Vector) was then applied on the slides, incubated for0 min, rinsed in TBS counterstained with Mayer’s hema-oxylin, dehydrated and mounted. Staining specificity wasscertained by omission of primary antisera or substitu-ion of primary antisera with non-immunized normal rabbiterum.

.3. Histomorphometric study

In order to determine the immunolabelled cell numbern granular convoluted tubules (GCTs), 12 randomly cho-en sections were averaged for each infected and controlroups. The GCT images were obtained at a final magnifica-ion of 400×, using a capture camera and microscope (Nikonclipse E200) interfaced with a personal computer and soft-are Image Tool. Immunolabelled cells were counted usinggrid with vertical and horizontal lines placed on a photomi-rography, with the aid of the confocal Assistant (Chapelill, USA). All cells located on the grid lines intersectionere counted.

.4. Serum testosterone levels

After submandibular glands removal, blood samples wereollected by cardiac puncture from the left ventricle. Serumas separated by centrifugation and stored at −20 ◦C.erum testosterone levels were measured using a commer-ially available radioimmunoassay kit (Testosterone Maiait/Biochem Immunosystems Italia S.P.A.) according to theanufacturer’s protocol.

.5. Statistical analysis

Data were expressed as mean ± S.D. (standard deviation).tatistical comparisons between groups were made usingNOVA analysis of variance followed by Student’s t-test orukey’s test. Significance was accepted when the p-value was0.05.

. Results

.1. Body weight

The body weight of the infected rats after 18 and 32 days

as significantly lower than that of controls (non-infected

ats). On the other hand, after 64 and 90 days post-infectionhe animals did not show significant differences when com-ared to controls (Fig. 1).

A. Moreira et al. / Tissue and Cell 40 (2008) 293–298 295

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Fig. 3. Sections of the submandibular glands were stained with H&E andthe numbers of granular convoluted cells counted. The results are expressedap

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ig. 1. Body weight of the rats during experimental Chagas’ disease. Ani-als were weighted 18, 32, 64 and 90 days after infection. The results are

xpressed as mean ± S.D. *p < 0.05 compared with non-infected animals athe same period.

.2. Histological studies

Our histological results confirmed the findings of Alvesnd Machado (1980). No evidence of inflammatory processas observed in the glands of infected animals even dur-

ng the acute phase of the disease. However, the patternf glandular development in the experimental animals wasonsiderably different from non-infected rats (Fig. 2A and). The main changes observed were acinar developmentnd delayed ductal maturation that was more evident in theeveloping granular ducts. After 90 days of infection thereas no difference between control and infected rats at theistological level (Fig. 2C and D, respectively).

.3. Histoquantitative analysis

The evolution of granular duct cells from the 18th to the0th day post-infection of control and infected rats are rep-

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ig. 2. Morphological aspect of the glandular convoluted tubules. (A) Submandibul8 days post-infection. It is possible to observe that the infected animals demonstndicated by arrowheads. (C) Submandibular glands of non-infected rats after 90 daere observed between control and infected rats after 90 days of infection. H&E st

s mean ± S.D. *p < 0.05 compared with non-infected animals at the sameeriod.

esented in Fig. 3. In the non-infected group, there was anncrease in the number of granular duct cells between 18 and0 days (294%; p < 0.0001) from 30.76 ± 7.71 on day 18 to9.70 ± 20.24 on day 90. Similarly, in the infected group,e observed an increase in the number of granular duct cells

anging between 13.60 ± 3.13 at day 18 and 84.10 ± 15.92n day 90 (618%; p < 0.0001). When comparing the infectednd non-infected rats, it was possible to observe a less num-er of granular cells in infected rats after 18 and 32 daysacute phase) compared to the control animals (p < 0.05),owever, after 64 and 90 days (chronic phase) the number

f granular cells in both groups was very similar (p > 0.05)Fig. 3).

ar glands of non-infected rats after 18 days. (B) Submandibular glands afterrated a decreased acinar development and a delayed ductal maturation asys. (D) Submandibular glands after 90 days post-infection. No differences

aining; bar = 10 �m.

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ig. 4. Circulating testosterone levels in control and infected rats after 18r 90 days of infection. *p < 0.05 compared with control group at the sameeriod of infection (Student’s t-test).

.4. Serum testosterone levels

Serum testosterone levels in infected animals after 18ays of infection were significantly lower than control group0.54 ± 0.09 vs. 0.03 ± 0.01 ng/ml, p < 0.05). After 90 daysf infection, no significant difference in testosterone levelsere observed between control group 5.26 ± 0.75 ng/ml and

nfected animals 4.58 ± 0.73 ng/ml (Fig. 4).

.5. Immunohistochemistry studies

Negative control staining was done by omitting exposureo the primary antibodies. Tissue staining specificity was con-rmed by lack of immunostaining for all experimental groupsdata not shown).

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ig. 5. Immunohistochemical photomicrographs expression of EGF from submandGF immunolabelled cells at the submandibular glands of non-infected after 18 d

mmunolabelled cells (B). Immunolabelling of EGF in non-infected (C) and infecte

ell 40 (2008) 293–298

EGF immunoreactivities were present mainly in theranular duct cells (Fig. 5). The glands of rats after 18ays of infection showed lower expression when com-ared with its respective control (Fig. 5A and B). However,t day 90, immunolabelling increased in the granularuct cells from infected rats showing a similar expres-ion pattern when compared to its respective controlFig. 5C and D).

. Discussion

In the present study, a significant reduction in testos-erone levels and the number of GCT after 18 days of T.ruzi inoculation was observed. However, after 90 days ofnfection, the numbers of GCT and hormone levels wereomparable to non-infected animals. Our results addition-lly demonstrated that body weight was negatively affectedy T. cruzi infection after 18 days, followed by bodyeight recuperation 90 days after inoculation, correlatingositively to EGF expression levels observed immunohis-ochemically. Since body weight has been reported to be

ore important than age to sexual maturation (Courout etl., 1970), our results suggest that rats infected with T. cruziay exhibit a delay in testes development with a reduc-

ion in testosterone levels. These alterations observed on thecute phase of experimental Chagas’ disease, would lead toetardation of granular cells differentiation and lower EGF

evels.

The submandibular glands of rodents are well known toe a major source of salivary EGF (Thulesen et al., 2002).CTs of adult rats, located between the intercalated and stri-

ibular glands of infected and non-infected rats after 18 and 90 days. Noteays (A), whereas submandibular of infected rats glands showed no EGF

d animals (D) after 90 days was similar. Magnification 400×; bar = 10 �m.

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ted portions of the duct system, consist of cells with largeecretory granules that contain a variety of bioactive peptides,ncluding growth factors such as EGF and NGF (Barka et al.,980; Gresik, 1994). EGF appears to be a potent factor in theaintenance of the integrity of the mucosal surface (Olsen et

l., 1984; Rao et al., 1997) as well as to regulate the chemi-al characteristics of the mucus coat (Sarosiek et al., 1988).he synthesis of salivary EGF seems to be affected in severalathophysiological conditions (Ohmura et al., 1987; Kelly etl., 1989; Calabro et al., 1990; Hormia et al., 1993; Rao etl., 1994; Helmrath et al., 1998). For instance, it has beeneported in parotid glands (Silva et al., 2000), that 18 daysf infection with T. cruzi, leads to a significant reduction inensity and volume of the acini and duct systems, as well assignificant increase in the amount of connective tissue and

ells undergoing mitosis. However, after 45 days, the glandsf infected rats returned to the normal pattern, suggestinghat in infected animals, the decrease in body weight coulde responsible for retarded sexual maturity, leading to lowerevels of testosterone and consequently decreasing the levelsf EGF and NGF.

The differentiation of GCT cells, which starts around 5eeks post-partum, as well as the maintenance of these cells

n adulthood, are under the control of the synergistic actionsf androgens, thyroid hormones and adrenocortical hormonesChretien, 1977; Gresik, 1994). Androgens are considered therimary factor in the differentiation of CGT. Surgical resec-ion of testis leads to a substantial regression of these tubules,hich is reversed by the administration of testosterone in

ats (Hiramatsu et al., 1994; Amano and Iseki, 1998). In theresent study, we observed that 18 days of infection may beufficient to introduce a decrease in testosterone levels, andlso histological changes in acinar development and delayeductal maturation. Furthermore, during the chronic phase90 days after inoculation) body weight recuperation occurslong with an increase in hormone levels, and morpholog-cal pattern recuperation of granular duct cells number andistological features. Since EGF is synthesized by GCT cellsnd based on the possibility that T. cruzi might infect salivarylands during the acute phase of the disease, we demonstratedhat the glandular synthesis of salivary EGF was affected byhagas’ disease.

Thus, within the limits of the present study, we may con-lude that the acute phase of Chagas’ disease developmentay cause a decrease in testosterone levels, and thereforedelay in GCT cells development and EGF production by

ubmandibular gland cells however, this alterations are sup-ressed after disease cronification.

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