effects of cholinergic stimulation with pyridostigmine bromide on chronic chagasic cardiomyopathic...

Research ArticleEffects of Cholinergic Stimulation with Pyridostigmine Bromideon Chronic Chagasic Cardiomyopathic Mice

Mariacutelia Beatriz de Cuba1 Marcus Paulo Ribeiro Machado1 Thais Soares Farnesi1

Angelica Cristina Alves1 Livia Alves Martins1 Lucas Felipe de Oliveira1

Caroline Santos Capitelli1 Camila Ferreira Leite1 Marcos Viniacutecius Silva1

Juliana Reis Machado1 Henrique Borges Kappel1 Helioswilton Sales de Campos1

Luciano Paiva1 Nataacutelia Lins da Silva Gomes2 Ana Carolina Guimaratildees Faleiros1

Constanccedila Felicia de Paoli de Carvalho Britto2 Wilson Savino2 Otaciacutelio Cruz Moreira2

Virmondes Rodrigues Jr1 Nicola Montano3 Eliane Lages-Silva1

Luis Eduardo Ramirez1 and Valdo Jose Dias da Silva1

1 Natural and Biological Sciences Institute Triangulo Mineiro Federal University Praca Manoel Terra 330 Centro38025-015 Uberaba MG Brazil

2 Oswaldo Cruz Institute 21040-900 Rio de Janeiro RJ Brazil3 Department of Clinical Sciences Internal Medicine II L Sacco Hospital University of Milan 20157 Milan Italy

Correspondence should be addressed to Valdo Jose Dias da Silva valdomednetcombr

Received 25 April 2014 Revised 16 July 2014 Accepted 20 July 2014 Published 24 August 2014

Academic Editor Marcelo T Bozza

Copyright copy 2014 Marılia Beatriz de Cuba et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

The aim of the present study was to assess the effects of an anticholinesterase agent pyridostigmine bromide (Pyrido) onexperimental chronic Chagas heart disease in mice To this end male C57BL6J mice noninfected (controlCon) or chronicallyinfected (5 months) with Trypanosoma cruzi (chagasicChg) were treated or not (NT) with Pyrido for one month At the endof this period electrocardiogram (ECG) cardiac autonomic function heart histopathology serum cytokines and the presenceof blood and tissue parasites by means of immunohistochemistry and PCR were assessed In NT-Chg mice significant changesin the electrocardiographic autonomic and cardiac histopathological profiles were observed confirming a chronic inflammatoryresponse Treatment with Pyrido in Chagasic mice caused a significant reduction of myocardial inflammatory infiltration fibrosisand hypertrophy which was accompanied by a decrease in serum levels of IFN120574with no change in IL-10 levels suggesting a shift ofimmune response toward an anti-inflammatory profile Lower nondifferent numbers of parasite DNA copies were observed in bothtreated and nontreated chagasic mice In conclusion our findings confirm themarked neuroimmunomodulatory role played by theparasympathetic autonomic nervous system in the evolution of the inflammatory-immune response toT cruzi during experimentalchronic Chagas heart disease in mice

1 Introduction

Chagas disease also known as American Trypanosomiasisan endemic parasitic disease caused by a flagellate protozoan(Trypanosoma cruzi) is highly prevalent throughout LatinAmerica Its major clinical manifestation is the chronic cha-gasic cardiomyopathy (CCC) which affects 13 of the chron-ically infected patients and may present severe symptomsand signs such as congestive heart failure thromboembolicphenomena cardiac arrhythmias and sudden death [1ndash4]

Pathological processes in the heart include mononuclearinflammatory infiltration focal myocarditis epicarditis andneuroganglionitis associated with variable focal fibrosis anda paucity of parasites which is poorly correlatedwithmyocar-dial inflammatory infiltration [2 4]

The pathogenesis of CCC includes the balance betweenparasite invasiveness and the host immune response par-ticularly the Th1Th2 balance which affects the resis-tancesusceptibility to T cruzi infection [5ndash7] Even though

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 475946 13 pageshttpdxdoiorg1011552014475946

2 Mediators of Inflammation

an imbalanced excessive production ofTh1 proinflammatorycytokines is critical to control of the parasite levels in bloodand tissue [8] this type of response can also be capable ofdestroying functional cardiomyocytes [8] and intracardiacautonomic neurons [2 4 9] This autonomic denervationinvolving mainly parasympathetic postganglionic neuronscauses a marked cardiac autonomic dysfunction [2 4 9ndash11]which may be involved in initiating life-threatening cardiacarrhythmias and sudden death [2 4] Curiously this vagalparasympathetic autonomic dysfunction is strongly associ-ated with inflammatory infiltration and immune activationnot only in Chagas heart disease but also in other types ofcardiopathy [12ndash15]

Although these data support a direct role of the immunesystem in mediating both cardiac and autonomic nervousdisturbances recent data indicate that changes in the auto-nomic nervous system also affect the pattern of immuneresponse and inflammation in several cardiac and noncardiacdisease states [15ndash17] including Chagas disease [18] Both thesympathetic and parasympathetic branches of the autonomicnervous system can exert substantial modulatory effects onthe immune response mainly by inhibiting the Th1 responseprofile [17 19ndash21] This new evidence also indicates thatcardiac sympathetic and particularly parasympathetic auto-nomic denervationdysfunction may also contribute to anincreased inflammatory response and possibly to enhancedparasite elimination [22] Data from our laboratory collectedin the context of acute Chagas disease in mice has recentlyconfirmed that cardiac autonomic denervationdysfunctionmight contribute to increased inflammation [18]

Taking into account the concepts described abovenew therapies based on manipulations of vagal neuroim-munomodulation of the heart may be beneficial for treatingheart diseases in general and chronic chagasic cardiomyopa-thy in particular [17 23] For example therapeutic approachesfor increasing cardiac vagal function thereby potentiating orstimulating the vagal anti-inflammatory reflex might havea positive impact on chronic chagasic cardiomyopathy Infact for other cardiopathies these strategies in both anexperimental and clinical context including chronic electricvagal stimulation [24] or pharmacological potentiation [25]have successfully improved cardiac outcomes

Pyridostigmine bromide an anticholinesterasic agentthat has been used for many years to treatmyasthenia gravisexhibits protective cardiovascular effects during short-termadministration that lead to a reduction of cardiovascular riskmarkers and an improvement of autonomic dysfunction [2626 27 27ndash29] By potentiating vagal parasympathetic func-tion this compound is thought to provoke sinus bradycardiato reduce AV nodal conduction and the refractory periodof action potentials and to increase the cardiac excitationthreshold among other direct cardiac effects It is alsobelieved that pyridostigmine bromide via potentiation of thecholinergic anti-inflammatory pathway causes a reductionin myocardial inflammation and fibrosis associated withan improvement in cardiac hypertrophy and remodelingHowever there have been no reports regarding the effectsof pyridostigmine bromide on the chronic phase of Chagasheart disease

Therefore the main aim of the present study was toevaluate the effects of cholinergic potentiation with the anti-cholinesterase agent pyridostigmine bromide on electrocar-diographical cardiac autonomic histological (inflammatoryinfiltration and fibrosis) immunological and parasitologicalparameters in C57BL6j mice infected with the Romildostrain of Trypanosoma cruzi during the chronic phase ofChagas heart disease

2 Methods

All experiments were performed onwild-type C57BL6jmiceobtained from the animal facility of the Department ofPhysiology of Triangulo Mineiro Federal University Uber-aba MG All animals were males weighed 20ndash30 g andwere maintained in the animal facility of the Departmentof Physiology at the Triangulo Mineiro Federal Universityon a rodent diet (Nuvilab CR1 Nuvital Nutrientes LtdaCuritiba PR Brazil) andwere givenwater ad libitum until thebeginning of the experimental protocols All the experimentswere carried out according to the ldquoPrinciples of LaboratoryAnimal Carerdquo formulated by the National Society forMedicalResearch England and ldquoThe Guide for the Care and Use ofLaboratory Animalsrdquo published by the US National Institutesof Health (NIH publication number 85-23 revised in 1996)All procedures were also submitted to and approved by theCommission for Ethics in the Use of Animals in Research ofthe Triangulo Mineiro Federal University

21 Parasite Inoculation To induce experimental Chagas dis-ease mice were intraperitoneally inoculated with 15000 try-pomastigote forms of the Romildo strain of T cruzi An addi-tional set of animals matched for gender and weight receivedintraperitoneal injections of the vehicle and these were des-ignated as the control noninfected group After inoculationall of the animals were observed at least twice daily tomonitor their general state and to assess mortality duringthe acute phase On the 12th day after inoculation levels ofparasitemia were measured by the microhematocrit methodfor the peripheral tail blood of all inoculated animals accord-ing to Brennerrsquos technique [30] to confirm infection Allsubsequent surgical procedures and experimental protocolswere performed at the 5th and 6th months of infectionduring the chronic phase of infection which is characterizedby light tissue invasion and low mortality

22 Experimental Groups The experimental groups weredivided according to the presence of Chagas disease and theadministration of pyridostigmine bromide during the monthfrom the 5th to the 6thmonth of observation (chronic phase)as described below The numbers of animals used in eachgroup are indicated in the Results section The followinggroups were investigated

Group ImdashCon-NT wild-type C57BL6j mice were inject-edwith vehicle as a control (Con) not treated (NT)with pyri-dostigmine bromide and evaluated after sixmonths at the endof the observation period

Mediators of Inflammation 3

Group IImdashCon-Pyrido wild-type C57BL6j were inject-ed with vehicle as a control (Con) and treated with 30mgkgpyridostigmine bromide (Pyrido) an anticholinesteraseagent dissolved in tap water for 30 days from the 5th to the6th month of observation

Group IIImdashChg-NTwild-typeC57BL6jmicewere inoc-ulated with 15000 trypomastigote forms of the Romildostrain of T cruzi (Chg) not treated (NT) with pyridostigminebromide and evaluated after six months at the end of theobservation period

Group IVmdashChg-Pyrido wild-type C57BL6j mice wereinoculated with 15000 trypomastigote forms of the Romildostrain of T cruzi (Chg) and treated with 30mgkg pyridostig-mine bromide (Pyrido) an anticholinesterase agent dis-solved in tap water for 30 days from the 5th to the 6th monthof observation

The solution drinking volume was monitored daily andthe pyridostigmine bromide concentration (approximately0084mgmL) was adjusted daily according to the drinkingvolume to maintain a daily mean ingested dose of 30mgkg

23 Conventional Electrocardiographic (ECG) Monitoring inAnesthetizedMice Immediately prior to inoculation (ECG1)at the 5th (ECG2 immediately prior to the pyridostigminebromide treatment) and at the 6th months of observation(ECG3 at the end of the experimental protocol prior to thesurgical procedures) all of the animals were submitted to aconventional ECG study under tribromoethanol (250mgkgip) anesthesia Needle electrodes were placed under theskin to record the conventional bipolar limb leads (I IIand III) the unipolar limb leads (aVR aVL and aVF) andthe unipolar precordial (chest) leads (VA the needle wasplaced immediately to the right of the sternum in the 4thintercostal space VB the needle was placed just to the left ofthe sternum in the 4th intercostal space and VC the needlewas positioned in the 5th intercostal space at the midaxillaryline) To avoid errors in the positioning of the leads thesame individual consistently placed the electrodes on theanimals The ECG was recorded using an ECG amplifier(model 8811A Hewlett-Packard Med Inst Waltham MAUSA) coupled to a 12-bit analogue-to-digital interface (DI-720-USB Dataq Instruments Inc Akron OH USA) at thesampling rate of 3 KHz on an IBM personal computer withthe analysis performed by Windaq-Pro+ software (DataqInstruments Inc Akron OH USA) The ECG was recordedfor 2 minutes The intervals and wavelengths (ms) werecalculated automatically using customized software followingwave identification and cursor placement The ECG tracingswere consistently analyzed by the same individual who wasblinded to the study protocolThe following ECG parameterswere examined (1) RR interval (RRi) (2) P wave duration(Pd) (3) PR interval (PR) (4) QRS duration (QRSd) (5) QTinterval (QT) and (6) corrected QT interval (cQT definedas the QT interval corrected for heart rate using Bazettrsquosequation where the corrected QTc was equal to QT (in s)RR(in s)12) In contrast to humans the T wave in small rodentsis not well characterized and appears as a shoulder of theQRS complex Accordingly to measure the QT interval we

used the apex of the T wave which can be determined withhigh accuracy The ECG parameters were determined fromeach lead and were averaged Additionally the presence ofcardiac arrhythmias and atrioventricular or intraventricularblockades among other alterations was analyzed by visualinspection of ECG tracings by a blinded ECG specialist

24 Long-Term Electrocardiographic Recordings in FreelyMoving Mice After the third ECG recording at the 6thmonth of observation the animals were reanesthetized withtribromoethanol (250mgkg ip) and a pair of stainless steelelectrodes were implanted inside the subcutaneous tissue tocollect chronic recordings of conventional bipolar limb ECGlead II The animals were also cannulated with polyethylenetubing placed in the jugular vein for drug administrationAfter the surgical procedures the animals were left to recoverin individual cages for at least 48ndash72 h

After 48ndash72 h of surgical recovery and in the absence ofanesthesia the electrodes were connected to an ECG ampli-fier (model 8811A Hewlett Packard Waltham MA USA)and the baseline ECG was sampled continuously (3 KHz) fora period of 30 minutes with a personal computer (IBMPC)equipped with a 12-bit analogue-to-digital interface (DI-720-USB Dataq Instruments Inc Akron OH USA) All animalswere always recorded between 800AM and 500 PM in aquiet condition and in a freelymoving stateThe time series ofRR intervals derived from these chronic ECG recordingswereused to study the cardiac autonomic modulation in heart ratevariability

25 Heart Rate Variability Analysis From the baseline chron-ic ECG recordings (30 minutes) the RR interval time serieswere derived automatically by the detection of the R wavepeaks using customized linear analysis software which waskindly provided by Dr Alberto Porta (University of MilanItaly) The time series of the RR intervals were dividedinto contiguous segments of 300 beats overlapping by half(Welch protocol) After calculating themean and variance foreach segment a model-based autoregressive spectral analysiswas performed as described elsewhere [31 32] Briefly amodel of the oscillatory components present in the stationarysegments of the beat-to-beat time series of the RR intervalswas calculated based on the Levinson-Durbin recursion andthe order for the model was chosen according to Akaikersquoscriterion [32] This procedure allows for the automatic quan-tification of the center frequency and power of each relevantoscillatory component in the time series The oscillatorycomponents were labeled as very low frequency (VLF) lowfrequency (LF) or high frequency (HF) when the central fre-quencies were within the bands of 001ndash010Hz 010ndash100Hzor 100ndash500Hz respectively [18]Thepower of the LF andHFcomponents of the heart rate variability was also expressedin normalized units which were obtained by calculating thepercentage of the LF and HF variability with respect to thetotal power after subtracting the power of the VLF compo-nent (frequencies lt 010Hz) The normalization proceduretends to minimize the effect of changes in the total power onthe absolute values of the LF and HF variabilities [31 32]

4 Mediators of Inflammation

26 Pharmacological Autonomic Blockade After 30 minutesof baseline chronic ECG recording half of the animals weregiven an intravenous injection of atropine sulfate (1mgkgiv) followed by an injection of propranolol (1mgkg iv)15 minutes later whereas the other half received injectionsin the reverse sequence (propranolol followed by atropinesulfate) This procedure allowed for the quantification of thecardiac parasympathetic and sympathetic autonomic effectsin the former and latter group respectively measured asthe differences between heart rate (HR) after atropine andbaseline HR (parasympathetic effect) or as the differencesbetween HR after propranolol and baseline HR (sympatheticeffect) as well as the measurement of intrinsic pacemakerheart rate (IHR) quantified as the heart rate after the doubleblockade with atropine sulfate followed by propranolol orpropranolol followed by atropine sulfate

27 Histopathological Examination At the end of the experi-mental protocol all animals were euthanized with an excessdose of sodium thiopental (100mgkg resp ip) and thechest cavity was then opened to remove the heart for histo-pathological analysis To evaluate the extent of inflammatoryinfiltration tissue damage fibrosis and parasite nests excisedhearts from all animals were cleaned in 09 saline solutionand fixed in phosphate-buffered 10 formalin solution for48 h

After embedding the samples in paraffin five 5ndash7 120583mthick longitudinal (four-chamber) sections of the heartswere stained with hematoxylin-eosin and analyzed using anupright lightmicroscope (Axiolab Carl Zeiss Inc Germany)All regions of the hearts were examined by two blindedobservers The inflammatory infiltration and parasite nestswere characterized using a semiquantitative approach and thescoring system described by Chapadeiro et al [33] A globalmyocardial inflammation score was defined for each animalas the sum of the scores from different regions of the heart

To quantify the fibrotic area in the myocardium con-tiguous longitudinal sections of the hearts were stained withPicrosirius Red which binds the collagen present in the tis-sue matrix and the slides were analyzed with polarized lightmicroscopy using KS300 software (Karl Zeiss Inc Ger-many)

To enhance the visualization of parasite nests or antigensin cardiac tissue an immunohistochemistry technique basedon the detection of the diaminobenzidine (DAB-) derivedchromogenwas performedThe chromogenwas generated bya secondary antibody labeled with peroxidase which bindsto a primary antibody against T cruzi antigens After anti-body labeling peroxidase reaction and costaining with hem-atoxylin slides were analyzed with an upright light micro-scope (Axiolab Carl Zeiss Inc Germany)

28 Quantification of Serum Levels of Cytokines At the endof the experimental protocol serum samples for each animalwere collected by means of cardiac puncture to performcytokine profiling (IFN-120574 TNF-120572 IL-2 IL-4 IL-5 and IL-10)via ELISA or the cytometric bead array (CBA) technique

Enzyme-Linked Immune Assay (ELISA) Serum concentra-tions of IFN-120574 and IL-10 weremeasured by ELISA using pairsof monoclonal antibodies in accordance with the manufac-turerrsquos specifications (BD Pharmingen) Briefly high-affin-ity 96-well plates (Nunc Roskilde Denmark) were sensitizedwith cytokine-specific monoclonal antibodies followed byblocking with PBS containing 2 BSA (Sigma) The seraand recombinant cytokines were then added and the plateswere incubated for 4 h at room temperature The plates werewashed and incubatedwith 1120583gmLbiotinylated anticytokinemonoclonal antibody at 37∘C for 2 h followed by washingand incubation with alkaline phosphatase-conjugated strep-tavidin at 37∘C for 2 hThe reactionwas developed using diso-dium-p-nitrophenyl phosphate (Sigma) in diethanolaminebuffer Absorbance was measured at 405 nm in a microplatereader (Bio-Rad 2550 Reader EIA CA USA) The cytokineconcentration was calculated using a linear regression anal-ysis of the absorbance values obtained for the recombinantcytokines and it was expressed as pgmL The sensitivity ofthe tests ranged from 2 to 20 pgmL

281 Flow Cytometry Using a Cytometric Bead Array (CBA)Measurement of the cytokines TNF-120572 and IL-2 (Th1 cytok-ines) and IL-4 and IL-5 (Th2 cytokines) in serum samples wasperformed using the cytometric bead array (CBA) kit (BDBiosciences USA) according to the manufacturerrsquos instruc-tions Briefly 50120583L of bead populations with discrete fluo-rescence intensities and coatedwith cytokine-specific captureantibodies was added to 50 120583L of mice sera and 50120583L of phy-coerythrin-conjugated anti-mouse Th1Th2 cytokine anti-bodies Simultaneously standards for each cytokine (0ndash5000 pgmL)weremixedwith cytokine capture beads and thephycoerythrin-conjugated reagent The vortexed mixtureswere incubated for 3 h Beads were washed and analyzedusing flow cytometry (FACSCalibur BD Biosciences USA)The quantity (pgmL) of each cytokine was calculated usingCellQuest software (BD Biosciences USA) Standard curveswere derived from the cytokine standards supplied with thekit The lower limit of detection ranged from 1 to 21 pgmLfor different cytokines

29 Parasite DNA Detection in the Blood and in the Heartwith Conventional PCR For parasite DNA isolation fromthe blood total blood samples of mice were collected intubes containing 6M guanidine HCl with 02M EDTA (pH8) (VV) DNA was extracted using the phenol-chloroform-isoamyl alcohol method according to Macedo et al [34]

For parasite DNA isolation from the heart half of thehearts excised at the end of the experimental protocol werehomogenized and DNA was extracted by the alkaline lysismethod

PCR specific for T cruzi was performed according toWincker et al [35] using the following primers to amplify afragment of 330 bp 121 (51015840-AAA TAA TGT ACG GG(GT)GAG ATG CAT GA-31015840) and 122 (51015840-GGT TCG ATT GGGGTT GGT GTA ATA TA-31015840) from a nonvariant region ofthe kinetoplast DNAminicircles of T cruziThe cycling con-ditions were as follows 95∘C for 5 minutes followed by 35

Mediators of Inflammation 5

Table 1 Electrocardiogram before treatment with pyridostigmine bromide Electrocardiographical parameters (expressed as mean plusmn SEM)collected after five months follow-up in noninoculated control (Con) or T cruzi-inoculated (Chg) anesthetized C57BL6j mice beforetreatment with pyridostigmine bromide (Pyrido) or vehicle (NT nontreated animals)

Con-NT Con-Pyrido Chg-NT Chg-Pyrido(119899 = 10) (119899 = 12) (119899 = 13) (119899 = 17)

RR (ms) 13973 plusmn 522 15083 plusmn 602 13904 plusmn 562 13335 plusmn 404Pd (ms) 1387 plusmn 150 138 plusmn 137 1856 plusmn 051lowast 1884 plusmn 072lowast

PR (ms) 3883 plusmn 261 3872 plusmn 228 4064 plusmn 098 4017 plusmn 129QRSd (ms) 146 plusmn 186 1361 plusmn 143 1753 plusmn 108lowast 1706 plusmn 100lowast

QT (ms) 2074 plusmn 226 1714 plusmn 160 2228 plusmn 096lowast 212 plusmn 101

cQT (ms12) 174 plusmn 051 142 plusmn 050 19 plusmn 008lowast 184 plusmn 009

RR RR interval Pd P wave duration PR PR interval QRSd QRS complex duration QT QT interval cQT corrected QT interval ms milliseconds lowast119875 lt005 versus Con-NT and

119875 lt 005 versus Con-Pyrido

Table 2 Electrocardiogram after treatment with pyridostigmine bromide Electrocardiographical parameters (expressed as mean plusmn SEM)collected at the 6thmonth of observation and after onemonth of treatment (from the 5th to 6thmonth of infection) in noninoculated control(Con) or T cruzi-inoculated (Chg) anesthetized C57BL6j mice treated with pyridostigmine bromide (Pyrido) or vehicle (NT nontreatedanimals)

Con-NT Con-Pyrido Chg-NT Chg-Pyrido(119899 = 10) (119899 = 12) (119899 = 10) (119899 = 17)

RR (ms) 13756 plusmn 337 14105 plusmn 538 15254 plusmn 501lowast 15298 plusmn 1293Pd (ms) 1401 plusmn 153 1338 plusmn 151 1868 plusmn 052lowastamp 1463 plusmn 053PR (ms) 3793 plusmn 196 4438 plusmn 251lowast 4545 plusmn 222lowast 432 plusmn 099QRSd (ms) 1445 plusmn 139 1347 plusmn 137 1504 plusmn 053 145 plusmn 041QT (ms) 1777 plusmn 187 1735 plusmn 138 2247 plusmn 104lowast 1821 plusmn 059cQT (ms12) 151 plusmn 051 149 plusmn 047 183 plusmn 009lowastamp 151 plusmn 006RR RR interval Pd P wave duration PR PR interval QRSd QRS complex duration QT QT interval cQT corrected QT interval lowast119875 lt 005 versus Con-NTms milliseconds amp119875 lt 005 versus Chg-Pyrido and

119875 lt 005 versus Con-Pyrido

cycles at 95∘C for 1 minute and 65∘C for 1 minute The pro-ducts of the reaction were revealed by electrophoresis ina 60 polyacrylamide gel and stained with silver nitratewhich was photographed with a digital camera

210 Statistical Analysis All numerical data are expressedas the means (plusmnSEM) whereas the semiquantitative datafrom the histological examinations are expressed as themedi-ans and the 25th and 75th percentiles According to the nor-mality and variance homogeneity of the distribution a para-metric statistic such as two-way ANOVA followed by Tukeyrsquosmultiple comparison test or a nonparametric test such astheMann-Whitney test was performed Categorical variableswere analyzed using the Fisher exact test All statistical calcu-lations were performed using SigmaStat 203 software (SPSSInc Chicago IL USA) The differences were consideredsignificant when 119875 lt 005

3 Results

Parasite detection in peripheral tail blood samples by themicrohematocrit technique performed on the 12th day afterinfection was positive for all T cruzi-inoculated animals andnegative for all control noninfected mice

Electrocardiographical parameters measured in the firstECG recording (ECG1) at the beginning of the experimen-tal protocol before T cruzi inoculation and treatment did

not show any significant difference among all experimentalgroups as expected However after five months of infectionand before the pyridostigmine bromide treatment chagasicanimals (from Chg-NT and Chg-Pyrido groups) presentedsignificant elongations of Pd QRSd QT and cQT (Table 1)indicating a global functional disturbance of heart

Interestingly as shown in Table 2 after one month of thepyridostigmine bromide treatment and six months of infec-tion Chg-Pyrido mice presented a significant reduction inPd (an atrial parameter) and cQT (a ventricular parameter)suggesting an improvement in the electrical function ofthe heart In contrast in Chg-NT mice most of the ECGparameters were significantly different compared to Con-NTmice It is worth noting that in the Con-Pyrido group asignificant increase in PR was observed as expected becausepyridostigmine bromide might be increasing vagal neuraltransmission in the atrioventricular node with a consequentreduction in the conduction velocity of the action potentialThese results suggest that pyridostigmine bromide treatmentcan be effective in improving electrical disorders induced bychronic chagasic cardiomyopathy

The results of heart rate variability analysis in time-(variance) and frequency-domain (spectral) parameters areshown in Table 3 A significant reduction in the absolutevalues of total variability (variance) and the spectral compo-nents VLF LF and HF was observed in Chg-NT mice com-pared toCon-NT andCon-PyridomiceThe LF andHF com-

6 Mediators of Inflammation

Table 3 Heart rate variability after treatment with pyridostigmine bromide Heart rate variability parameters (expressed as mean plusmn SEM)collected at the 6thmonth of observation and after onemonth of treatment (from the 5th to 6thmonth of infection) in noninoculated control(Con) or T cruzi-inoculated (Chg) freely moving C57BL6j mice treated with pyridostigmine bromide (Pyrido) or vehicle (NT nontreatedanimals)

Con-NT Con-Pyrido Chg-NT Chg-Pyrido(119899 = 9) (119899 = 9) (119899 = 9) (119899 = 12)

RR (ms) 11797 plusmn 397 11267 plusmn 414 10427 plusmn 432 11374 plusmn 433HR (bpm) 51681 plusmn 835 54357 plusmn 906 59061 plusmn 982 53907 plusmn 876Variance (ms2) 21861 plusmn 1235 14896 plusmn 991 3213 plusmn 512lowastamp 6357 plusmn 611VLF (ms2) 10606 plusmn 893 6937 plusmn 723 1176 plusmn 284lowastamp 2260 plusmn 390LF (ms2) 5784 plusmn 628 5231 plusmn 623 1110 plusmn 377lowast 2199 plusmn 509LF (nu) 5183 plusmn 413 5624 plusmn 409 4780 plusmn 457 3925 plusmn 491HF (ms2) 5471 plusmn 686 2719 plusmn 444 901 plusmn 319lowastamp 1878 plusmn 353HF (nu) 4754 plusmn 411 4180 plusmn 397 4805 plusmn 449 5870 plusmn 486LFHF 231 plusmn 153 404 plusmn 148 260 plusmn 154 292 plusmn 201RR RR interval HR heart rate VLF very low frequency spectral component LF low frequency spectral component HF high frequency spectral componentms milliseconds bpm beats per minute nu normalized units lowast119875 lt 005 versus Con-NT and amp

119875 lt 005 versus Chg-Pyrido

ponents expressed in normalized units and the LFHF ratiowere not different from Con-NT mice In contrast chagasicmice treated with pyridostigmine bromide (the Chg-Pyridogroup) presented values of variance and VLF andHF spectralcomponents that were significantly higher compared to Chg-NT mice suggesting an improvement of some autonomicparameters that changed during chronic chagasic cardiomy-opathy (Table 3)

The autonomic dysfunction observed in the heart ratevariability analysis of Chg-NT mice and its improvementin Chg-Pyrido mice was confirmed by pharmacologicalautonomic blockade with atropine sulfate or propranololTheuse of these autonomic blockers showed that vagal para-sympathetic effects significantly decreased without a changein sympathetic effects in Chg-NT mice compared with Con-NT mice (Figure 1) Chagasic mice treated with pyridostig-mine bromide showed values of vagal parasympathetic effectsthat were significantly higher compared to Chg-NT miceand similar to those found in Con-NT mice (Figure 1)Additionally noninfected control mice treated with pyri-dostigmine bromide showed vagal parasympathetic effectsthat markedly increased without any changes in sympatheticeffects compared with Con-NT mice (Figure 1) No changesin the intrinsic pacemaker heart rate (IHR) were found in anyof the experimental groups after the six-month observationperiod

The relative cardiac weight of Chg-NT mice was sig-nificantly higher than that observed in the other groups(Figure 2) indicating that cardiac hypertrophy was inducedby Chagas disease in mice and that pyridostigmine bromidetreatment was able to reverse or impair the development ofcardiac hypertrophy in chagasic mice

A semiquantitative examination of cardiac inflammatoryinfiltration indicated discrete to mild diffuse myocarditis(average inflammatory score was equal to 05 11987525 = 05and 11987575 = 05 119875 lt 00001 versus Con-NT) which wasobserved in the atria (Figures 3(g) and 3(h)) the ventricles(Figures 3(e) and 3(f)) and the neural ganglia (Figure 3(g))in the CHG-NT mice in comparison to normal CON-NTmice (Figures 3(a) 3(b) 3(c) and 3(d)) which presented no

inflammatory infiltration (average inflammatory score wasequal to 00 11987525 = 00 and 11987575 = 00) In Chg-Pyrido mice reduced inflammatory infiltration which wascategorized as very discrete to discrete (average inflammatoryscore was equal to 0125 11987525 = 00 and 11987575 = 025119875 lt 0001 versus Chg-NT) was observed in the atria theventricles (Figures 3(i) 3(j) 3(k) and 3(l)) and the neuralganglia As expected in this mouse model of chronic Chagasdisease no amastigote nests in the myocardium were foundin either Chg-NT or Chg-Pyrido mice

In different sections of the heart stained by PicrosiriusRed the morphometry of fibrosis revealed a marked and dif-fuse increase in the fibrotic area in both absolute (120583m2)and relative () values in the Chg-NT group comparedto Chg-NT mice (Figure 4) Interestingly Chg-Pyrido micepresented significantly reduced fibrotic areas in all cardiacchambers (Figure 4) compared to Chg-NT mice Despitethese reduced values the fibrotic areas were still larger thanthose found in Con-NT mice (Figure 4)

The serum levels of the cytokines IL-2 IL-4 IL-5 andTNF-120572 measured with the CBA technique did not reveal anychange in any of the experimental groups In fact the serumlevels of IL-2 and IL-4 were undetectable with values near0 pgmL However using the ELISA technique serum levelsof IFN-120574were significantly higher in Chg-NTmice comparedto Chg-Pyrido mice (119875 lt 005) No IFN-120574 was detected inCon-NT mice Serum levels of IL-10 measured with ELISAtechnique were significantly higher in both Chg-NT andChg-Pyrido mice compared to Con-NT mice (119875 lt 005)No differences were found in the serum IL-10 levels betweenChg-NT and Chg-Pyrido mice (Figure 5)

The immunohistochemical technique using peroxidase tolabel T cruzi parasite nests or antigens revealed a completeabsence of parasites in control noninfected mice whereasT cruzi labeling was positive for 6250 of Chg-NT and8181 of Chg-Pyrido mice (119875 = 0603 not significant usingthe Fisher exact test) The parasite antigens were diffuselydistributed throughout the heart tissue (Figure 6)

Using a conventional PCRmethod to detect parasiteDNAin blood andheart during the chronic phase ofChagas disease

Mediators of Inflammation 7

0

minus50

minus100

minus150

ΔH

R (b

pm)

Con-NT Con-Pyrido Chg-NT Chg-Pyrido

(a)

Con-NT Con-Pyrido Chg-NT Chg-Pyrido0

80

60

40

20

100

ΔH

R (b

pm) amp

lowast

lowast

(b)

Figure 1Heart rate responses to pharmacological autonomic blockadeHeart rate responses (ΔHR) to propranolol (sympathetic effect) (Panel(a)) or to atropine sulfate (vagal parasympathetic effect) (Panel (b)) expressed asmeanplusmn SEM andmeasured at the 6thmonth of observationand after one month of treatment (from the 5th to 6th month of infection) in noninoculated control (Con) or in T cruzi-inoculated (Chg)freely moving C57BL6j mice treated with pyridostigmine bromide (Pyrido) or vehicle (NT nontreated animals) (lowast119875 lt 005 versus Con-NTamp119875 lt 005 versus Chg-NT)

Relat

ive c

ardi

ac w

eigh

t (m

gg)

0

2

4

6

8

10

Con-NT Con-Pyrido Chg-NT Chg-Pyrido

amp

lowast

Figure 2 Relative cardiac weights after treatment with pyridostig-mine bromide Relative cardiac weights (in mgg) expressed asmean plusmn SEM calculated at the 6th month of observation and afterone month of treatment (from the 5th to 6th month of infection)in noninoculated control (Con) or in T cruzi-inoculated (Chg)C57BL6j mice treated with pyridostigmine bromide (Pyrido) orvehicle (NT non treated animals) (lowast119875 lt 005 versus Con-NT andamp119875 lt 005 versus Chg-NT)

in mice a complete absence of parasite DNA in noninfectedcontrol mice was observed whereas T cruziDNA was foundin 1667 and 429 (119875 = 0559) in the blood of Chg-NTandChg-Pyridomice respectively and in 7273 and 8667(119875 = 0614) of heart samples from Chg-NT and Chg-Pyridomice respectively

4 Discussion

To our knowledge the present study is the first to analyze theeffects of the deliberate potentiation of cholinergic signalingusing pyridostigmine bromide an anticholinesterase agentduring the chronic phase of experimental Chagas diseasein mice Furthermore this study evaluated the effects ofpyridostigmine bromide treatment on electrocardiographicautonomic histopathological immunoinflammatory andparasitological parameters of Chagas disease

Thepresent study is to our knowledge the first to demon-strate autonomic dysfunction induced by chronic Chagasdisease in an experimental mouse model This dysfunctionwas characterized by a marked reduction in heart ratevariability with reduced variance (time-domain parameter)and the VLF LF and HF components (frequency-domainparameters) of heart rate variability as well as a concurrentreduction in the cardiac vagal effect without an apparentchange in sympathetic effects as measured by pharmacolog-ical blockade in chronic control chagasic animals Only mor-phological reports describing the ganglia and nerve lesions inchagasic mice have been published in the literature [36]Thisfinding follows our previous report of a similar autonomicdysfunction inmicewith acuteChagas disease [18]This auto-nomic dysfunctionwas accompanied by electrocardiographicchanges associated with mild diffuse inflammatory infiltra-tion of mononuclear cells as well as fibrosis and hypertrophyin the atrial and ventricular myocardium and the elevationof IFN-120574 and IL-10 in the blood serum of wild-type anduntreated chagasic C57BL6j mice which confirms the pres-ence of chronic myocarditis six months following infectionwith T cruzi This increase in serum levels of IFN-120574 and IL-10 in chronic chagasic mice matches similar previous reportsin human beings [37ndash39] Additionally parasite antigens andDNA were detected by immunohistochemical labeling in theheart and by PCR in blood and heart respectively reinforcingthe role played by parasites in inducing andor maintainingchronic infection Because our findings are consistent withprevious observations in hamsters rabbits dogs and humansduring the chronic phase of Chagas disease [10 40] themouse model used here appears to represent a suitable toolfor future experimental studies in Chagas heart disease

During the chronic phase of Chagas heart disease thisreport revealed a significant increase in cardiac vagal para-sympathetic autonomic modulation associated with a signifi-cant reduction of inflammatory infiltration in the myocar-dium of infected mice treated with pyridostigmine bromideduring the last month of a six-month observation period

8 Mediators of Inflammation

(e) (f)

(g) (h)

(i) (j)

(k) (l)

(b)(a)

(c) (d)

Figure 3 Cardiac histopathology after treatment with pyridostigmine bromide Photomicrographs of cardiac sections stained with H-EPanels (a) and (b) ventricular sections from a control nontreated mouse Panels (c) and (d) ventricular sections from a pyridostigminebromide-treated control mouse Panels (e) and (f) ventricular sections from a chagasic nontreated mouse Panels (g) and (h) atrial sectionsfrom a nontreated chagasic mouse showing respectively a ganglionitis and neuritis focus Panels (i) (j) (k) and (l) different ventricularsections from a chagasic mouse treated with pyridostigmine bromide Note the lower inflammatory infiltration into heart tissues from achagasic mouse treated with pyridostigmine bromide (Left panels magnification = 100x right panels magnification = 200x)

Mediators of Inflammation 9

0

500

1000

1500

Con-NT Con-Pyrido Chg-NT Chg-Pyrido

ampFi

bros

is ar

ea (120583

m2)

lowast

(a)

0

500

1000

1500

2000

Con-NT Con-Pyrido Chg-NT Chg-Pyrido

Fibr

osis

area

(120583m

2)

amplowast

(b)

0

500

1000

1500

2000

Con-NT Con-Pyrido Chg-NT Chg-Pyrido

Fibr

osis

area

(120583m

2)

amplowast

(c)

0

500

1000

1500

2000

2500

Con-NT Con-Pyrido Chg-NT Chg-Pyrido

Fibr

osis

area

(120583m

2)

amplowast

(d)

0

500

1000

1500

Con-NT Con-Pyrido Chg-NT Chg-Pyrido

Fibr

osis

area

(120583m

2)

amplowast

(e)

Figure 4 Cardiac fibrosis after treatment with pyridostigmine bromide Fibrotic areas (in 120583m2) expressed as mean plusmn SEM for the right(Panel (a)) and left (Panel (b)) atria the right (Panel (c)) and left (Panel (d)) ventricles and the atrioventricular septum (Panel (e)) measuredafter Picrosirius Red staining at the 6th month of observation and after one month of treatment (from the 5th to 6th month of infection) innoninoculated control (Con) or in T cruzi-inoculated (Chg) C57BL6j mice treated with pyridostigmine bromide (Pyrido) or vehicle (NTnontreated animals) (lowast119875 lt 005 versus Con-NT amp119875 lt 005 versus Chg-Pyrido and

119875 lt 005 versus Con-Pyrido)

Because pyridostigmine bromide is an anticholinesterasicagent which increases the bioavailability of acetylcholine inthe synaptic varicosities the increase in cardiac vagal para-sympathetic autonomic function demonstrated by the higherheart rate variability and cardiac vagal effect was expectedand was verified after treatment This vagal stimulation effectof pyridostigmine bromide confirms previous reports inother experimental contexts [41]

The anti-inflammatory effect of pyridostigmine bromidewas associatedwith amarked reduction ofmyocardial fibrosisand hypertrophy Serum levels of IFN-120574 also decreasedwhereas TNA-120572 trended to decrease both of which are Th1proinflammatory cytokines without any change in the aug-mented serum levels of IL-10

The combined analysis of these results suggests that pyri-dostigmine bromide may act on the heart at least partially

10 Mediators of Inflammation

0

1

2

3

Con-NT Con-Pyrido Chg-NT Chg-Pyrido

TNF-120572

(pg

mL)

(a)

IL-5

(pg

mL)

00

05

10

15

20

25

Con-NT Con-Pyrido Chg-NT Chg-Pyrido

(b)

0

100

200

300

Con-NT Con-Pyrido Chg-NT Chg-Pyrido

IFN

-120574(p

gm

L)

amplowast

(c)

IL-1

0 (p

gm

L)

0

100

200

300

400

Con-NT Con-Pyrido Chg-NT Chg-Pyrido

lowast

(d)

Figure 5 Serum cytokines after treatment with pyridostigmine bromide Serum levels (in pgmL) expressed as mean plusmn SEM of tumornecrosis factor-120572 (TNF-120572 Panel (a)) interleukin-5 (IL-5 Panel (b)) measured by the cytometric bead array technique interferon-120574 (IFN-120574Panel (c)) and interleukin-10 (IL-10 Panel (d)) measured by ELISA at the 6th month of observation and after one month of treatment (fromthe 5th to 6th month of infection) in noninoculated control (Con) or T cruzi-inoculated (Chg) C57BL6j mice treated or not (NT) withpyridostigmine bromide (Pyrido) (lowast119875 lt 005 versus Con-NT amp119875 lt 005 versus Chg-Pyrido and

119875 lt 005 versus Con-Pyrido)

increasing the vagal parasympathetic activity via the accu-mulation of acetylcholine in themyocardiumThis accumula-tion was confirmed by improvements in heart rate variabilityand vagal parasympathetic effect which acting on immunecells may exert immunomodulatory effects thereby shiftingthe immune response toward the predominance of an anti-inflammatory response

These new findings in the chronic Chagas disease contextreinforce the idea of an intimate interplay between theimmune and autonomic nervous systems and the potentialuse of parasympathetic stimulation to treat chagasic myocar-ditis [18 23] Additionally the improvement of cardiacinflammation fibrosis and hypertrophy in pyridostigminebromide-treated chronic chagasic animals suggests that thedecrease of vagal autonomic function due to ganglionic neu-ronal lesions and denervation which occur precociously dur-ing the acute phase may play an important immunomodula-tory role in the increased Th1 immune response and inflam-matory infiltration as verified during the chronic phase of thedisease

Even though our results support the immunomodulatoryrole played by the cholinergic vagal parasympathetic nervoussystem in the heart we cannot rule out a possible roleof pyridostigmine bromide in increasing local acetylcholinelevels via its release from nonneural local structures suchas endothelial cells cholinergic lymphocytes and cardiomy-ocytes [42] In fact these nonneural sources of acetyl-choline potentiated by pyridostigmine bromide treatmentmay explain the substantial improvement in ECG abnormal-ities inflammation fibrosis and hypertrophy observed inboth ventricles of the heart which are poorly innervated byvagal parasympathetic fibers

Data from the immunohistochemical labeling of T cruziantigens and T cruzi DNA detection by PCR showed thatparasites are present in the heart tissue and blood of thechagasicmice sixmonths after infection confirming previousreports [38 43] The number of animals positive for parasiteantigens or DNA did not differ between pyridostigminebromide-treated and nontreated chagasic animals The per-sistence of the parasite in the chronic phase even at a very

Mediators of Inflammation 11

100120583m

(a)

100120583m

(b)

100120583m

(c)

40120583m

(d)

40120583m

(e)

40120583m

(f)

Figure 6 Immunohistochemistry for parasite antigens Photomicrographs from left ventricular sections stained with T cruzi antigens byperoxidase reaction Panels (a) and (d) control nontreated mouse Panels (b) and (e) chagasic nontreated mouse Panels (c) and (f) chagasicmouse treated with pyridostigmine bromide Top panels magnification = 100x bottom panels magnification = 200x Note the gray colorlabeling T cruzi antigens throughout the sections in Panels (b) (c) (e) and (f)

low level seems to play an important role in the pathogenesisof chronic Chagas disease because the low parasite loadmay continuously stimulate an immune response [38 43]Although antigens or DNA from T cruzi were detected inthe present study no parasite pseudocysts or amastigoteforms were observed in the heart tissue in either treated ornontreated chagasic mice This lack of entire parasites at thesite of cardiac lesions suggests the possibility of a remoteniche for the parasites such as smooth muscle cells adipo-cytes or skeletal muscle cells [43]

In conclusion our results support the notion that auto-nomic dysfunction is a primary cause of the pro-anti-inflam-matory imbalance observed in inflammatory diseases favor-ing the shift of immune response toward the predominance ofproinflammatory response thereby contributing to the path-ogenesis of these diseases in general and Chagas heart diseasein particular Additionally our findings showed the potential

beneficial effects of anticholinesterasic agents particularlypyridostigmine bromide in increasing cardiac autonomicmodulation and reducing inflammatory response to theheart

Conflict of Interests

The authors declare no conflicts of Interests

Authorsrsquo Contribution

Marilia Beatriz de Cuba and Marcus Paulo Ribeiro Machadoequally contributed to this study

Acknowledgments

This study was supported by the Ministero dellrsquoIstruzioneUniversitarsquo e della Ricerca Scientifica PRIN 2007 Grant to

12 Mediators of Inflammation

Nicola Montano and a Research Fellowship Grant (Procnumber 3080162009-2) from the ConselhoNacional deDes-envolvimento Cientifico e Technologico (CNPq)-Brazil toValdo Jose Dias da Silva

References

[1] C J Chagas ldquoAmerican trypanosomiasisrdquo Memorias do Insti-tuto Oswaldo Cruz vol 3 pp 219ndash275 1911

[2] A Prata ldquoClinical and epidemiological aspects of Chagas dis-easerdquo The Lancet Infectious Diseases vol 1 no 2 pp 92ndash1002001

[3] J R Coura and P A Vıas ldquoChagas disease a new worldwidechallengerdquo Nature vol 465 no 7301 pp S6ndashS7 2010

[4] J A Marin-Neto E Cunha-Neto B C Maciel and M VSimoes ldquoPathogenesis of chronic Chagas heart diseaserdquo Circu-lation vol 115 no 9 pp 1109ndash1123 2007

[5] S A Morris H B Tanowitz M Wittner and J P BilezikianldquoPathophysiological insights into the cardiomyopathy of Cha-gasrsquo diseaserdquo Circulation vol 82 no 6 pp 1900ndash1909 1990

[6] M J M Alves and R A Mortara ldquoA century of research Whathave we learned about the interaction of Trypanosoma cruziwith host cellsrdquoMemorias do Instituto Oswaldo Cruz vol 104supplement 1 pp 76ndash88 2009

[7] C A Hunter L A Ellis-Neyes T Slifer et al ldquoIL-10 is requiredto prevent immune hyperactivity during infection with Try-panosoma cruzirdquoThe Journal of Immunology vol 158 no 7 pp3311ndash3316 1997

[8] A R L Teixeira MM Hecht M C Guimaro A O Sousa andN Nitz ldquoPathogenesis of Chagasrsquo disease parasite persistenceand autoimmunityrdquo Clinical Microbiology Reviews vol 24 no3 pp 592ndash630 2011

[9] L E Ramirez E Lages-Silva JM Soares Jr and E ChapadeiroldquoExperimental hamster infection by Trypanosoma cruzi thechronic phaserdquo Revista da Sociedade Brasileira de MedicinaTropical vol 26 no 4 pp 253ndash254 1993

[10] V J D da Silva M P R Machado A M Rocha R M Padilhaand L E Ramirez ldquoAnalysis of cardiac autonomic function inhamsters with Chagas diseaserdquo Revista da Sociedade Brasileirade Medicina Tropical vol 36 supplement 2 pp 11ndash12 2003

[11] A R Perez S D Silva-Barbosa L R Berbert et al ldquoImmuno-neuroendocrine alterations in patients with progressive formsof chronic Chagas diseaserdquo Journal of Neuroimmunology vol235 no 1-2 pp 84ndash90 2011

[12] R E Kleiger J P Miller J T Bigger Jr and A J MossldquoDecreased heart rate variability and its association withincreased mortality after acute myocardial infarctionrdquo TheAmerican Journal of Cardiology vol 59 no 4 pp 256ndash262 1987

[13] A Hamaad M Sosin A D Blann J Patel G Y H Lip andR J MacFadyen ldquoMarkers of inflammation in acute coro-nary syndromes association with increased heart rate andreductions in heart rate variabilityrdquo Clinical Cardiology vol 28no 12 pp 570ndash576 2005

[14] P J Hauptman P J Schwartz M R Gold et al ldquoRationale andstudy design of the INcrease of Vagal TonE in Heart Failurestudy INOVATE-HFrdquoTheAmerican Heart Journal vol 163 no6 pp 954ndash962 2012

[15] V Papaioannou I Pneumatikos and N Maglaveras ldquoAssoci-ation of heart rate variability and inflammatory response inpatients with cardiovascular diseases current strengths andlimitationsrdquo Front Physiol vol 4 article 174 2013

[16] D J vanWesterloo I A J Giebelen S Florquin et al ldquoThe cho-linergic anti-inflammatory pathway regulates the host responseduring septic peritonitisrdquo Journal of Infectious Diseases vol 191no 12 pp 2138ndash2148 2005

[17] K J Tracey ldquoReflex control of immunityrdquo Nature ReviewsImmunology vol 9 no 6 pp 418ndash428 2009

[18] M P R Machado A M Rocha L F de Oliveira et al ldquoAuto-nomic nervous system modulation affects the inflammatoryimmune response in mice with acute Chagas diseaserdquo Experi-mental Physiology vol 97 no 11 pp 1186ndash1202 2012

[19] V M Sanders ldquoThe role of norepinephrine and beta-2-adren-ergic receptor stimulation in the modulation of TH1 TH2 andB lymphocyte functionrdquo Advances in Experimental Medicineand Biology vol 437 pp 269ndash278 1998

[20] I J Elenkov and G P Chrousos ldquoStress hormones Th1Th2patterns proanti-inflammatory cytokines and susceptibility todiseaserdquo Trends in Endocrinology and Metabolism vol 10 no 9pp 359ndash368 1999

[21] L V Borovikova S IvanovaM Zhang et al ldquoVagus nerve stim-ulation attenuates the systemic inflammatory response to endo-toxinrdquo Nature vol 405 no 6785 pp 458ndash462 2000

[22] E E Benarroch ldquoAutonomic-mediated immunomodulationand potential clinical relevancerdquo Neurology vol 73 no 3 pp236ndash242 2009

[23] M P R Machado and V J D da Silva ldquoAutonomic neuroim-munomodulation in chagasic cardiomyopathyrdquo ExperimentalPhysiology vol 97 no 11 pp 1151ndash1160 2012

[24] M Li C Zheng T Sato T Kawada M Sugimachi and KSunagawa ldquoVagal nerve stimulation markedly improves long-term survival after chronic heart failure in ratsrdquoCirculation vol109 no 1 pp 120ndash124 2004

[25] Y Okazaki C Zheng M Li and M Sugimachi ldquoEffect of thecholinesterase inhibitor donepezil on cardiac remodeling andautonomic balance in rats with heart failurerdquo The Journal ofPhysiological Sciences vol 60 no 1 pp 67ndash74 2010

[26] S M Serra R V Costa R R Teixeira de Castro S S Xavierand A C Lucas daNobrega ldquoCholinergic stimulation improvesautonomic and hemodynamic profile during dynamic exercisein patients with heart failurerdquo Journal of Cardiac Failure vol 15no 2 pp 124ndash129 2009

[27] L I Zimerman A Liberman R R T Castro J P Ribeiroand A C L Nobrega ldquoAcute electrophysiologic consequencesof pyridostigmine inhibition of cholinesterase in humansrdquoBrazilian Journal of Medical and Biological Research vol 43 no2 pp 211ndash216 2010

[28] R R T Castro G Porphirio S M Serra and A C L NobregaldquoCholinergic stimulation with pyridostigmine protects againstexercise induced myocardial ischaemiardquo Heart vol 90 no 10pp 1119ndash1123 2004

[29] R R T Castro SM Serra G Porphirio F S N SMendes L PJ Oliveira and A C L Nobrega ldquoPyridostigmine reduces QTcinterval during recovery from maximal exercise in ischemicheart diseaserdquo International Journal of Cardiology vol 107 no1 pp 138ndash139 2006

[30] Z Brener ldquoTherapeutic activity and criterion of cure on miceexperimentally infected with Trypanosoma cruzirdquo Revista doInstituto de Medicina Tropical de Sao Paulo vol 4 pp 389ndash3961962

[31] Task Force of the European Society of Cardiology and theNorth American Society of Pacing and ElectrophysiologyldquoHeart rate variability standards ofmeasurement physiological

Mediators of Inflammation 13

interpretation and clinical userdquo Circulation vol 93 pp 1043ndash1065 1996

[32] N Montano A Porta C Cogliati et al ldquoHeart rate variabilityexplored in the frequency domain a tool to investigate the linkbetween heart and behaviorrdquo Neuroscience and BiobehavioralReviews vol 33 no 2 pp 71ndash80 2009

[33] E Chapadeiro P S Beraldo P C Jesus W P Oliveira Juniorand L F Junqueira Junior ldquoCardiac lesions inWistar rats inocu-lated with various strains of Trypanosoma cruzirdquo Revista daSociedade Brasileira de Medicina Tropical vol 21 no 3 pp 95ndash103 1988

[34] A M Macedo M S Martins E Chiari and S D J PenaldquoDNAfingerprinting ofTrypanosoma cruzi a new tool for char-acterization of strains and clonesrdquo Molecular and BiochemicalParasitology vol 55 no 1-2 pp 147ndash153 1992

[35] P Wincker M Bosseno C Britto et al ldquoHigh correlationbetween Chagasrsquo disease serology and PCR-based detection ofTrypanosoma cruzi kinetoplast DNA in Bolivian children livingin an endemic areardquo FEMS Microbiology Letters vol 124 no 3pp 419ndash423 1994

[36] L CV Ribeiro AA Barbosa Jr andZAAndrade ldquoPathologyof intracardiac nerves in experimental chagas diseaserdquo Memo-rias do Instituto Oswaldo Cruz vol 97 no 7 pp 1019ndash1025 2002

[37] R C Ferreira B M Ianni L C J Abel et al ldquoIncreased plasmalevels of tumor necrosis factor-alpha in asymptomaticldquoinde-terminaterdquo and Chagas disease cardiomyopathy patientsrdquoMemorias do Instituto Oswaldo Cruz vol 98 no 3 pp 407ndash4112003

[38] F R S Gutierrez P M M Guedes R T Gazzinelli and J SSilva ldquoThe role of parasite persistence in pathogenesis of chagasheart diseaserdquo Parasite Immunology vol 31 no 11 pp 673ndash6852009

[39] A S de Melo V M B de Lorena S C de Moura Braz CDocena and Y de Miranda Gomes ldquoIL-10 and IFN-120574 geneexpression in chronic Chagas disease patients after in vitrostimulation with recombinant antigens of Trypanosoma cruzirdquoCytokine vol 58 no 2 pp 207ndash212 2012

[40] M B Soares and R R dos Santos ldquoImmunopathology ofcardiomyopathy in the experimental Chagas diseaserdquoMemoriasdo Instituto Oswaldo Cruz vol 94 supplement 1 pp 257ndash2621999

[41] R N de la Fuente B Rodrigues I C Moraes-Silva et alldquoCholinergic stimulation with pyridostigmine improves auto-nomic function in infarcted ratsrdquo Clinical and ExperimentalPharmacology and Physiology vol 40 no 9 pp 610ndash616 2013

[42] C Rocha-Resende A Roy R Resende et al ldquoNon-neuronalcholinergicmachinery present in cardiomyocytes offsets hyper-trophic signalsrdquo Journal of Molecular and Cellular Cardiologyvol 53 no 2 pp 206ndash216 2012

[43] F Nagajyothi F S Machado B A Burleigh et al ldquoMechanismsof Trypanosoma cruzi persistence in Chagas diseaserdquo CellularMicrobiology vol 14 no 5 pp 634ndash643 2012

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