ORIGINAL ARTICLE

Pentoxifylline reduces inflammation

and prevents myocardial perfusion
derangements in experimental chronic Chagas’
cardiomyopathy
Denise Mayumi Tanaka, PhD,a Camila Godoy Fabricio, MS,a José A. Marin-Neto,
PhD,a Antônio Carlos Leite de Barros Filho, MS,a Luciano Fonseca Lemos de
Oliveira, PhD,b Jorge Mejia, PhD,c Rafael Ribeiro Almeida, PhD,d Raquel de
Souza Vieira, MS,d Carla Duque Lopes, PhD,a Sabrina Setembre Batah, MS,a
Henrique Turin Moreira, PhD,a Maria de Lourdes Higuchi, PhD,d
Edecio Cunha Neto, PhD,d Alexandre Todorovic Fabro, PhD,a
Stephan G. Nekolla, PhD,e Minna Moreira Dias Romano, PhD,a and
Marcus Vinı́cius Simões, PhDa,f
a
Medical School of Ribeirao Preto, University of São Paulo, Sao Paulo, Brazil
b
Department of Applied Physical Therapy, Institute of Health Sciences, Federal University of
Triangulo Mineiro, Uberaba, Brazil
c
Hospital Israelita Albert Einstein, Sao Paulo, Brazil
d
Faculty of Medicine, Heart Institute (InCor), University of Sao Paulo, Sao Paulo, Brazil
e
Department of Nuclear Medicine at Technische Universität, Munich, Germany
f
Cardiology Division, Internal Medicine Department, Hospital das Clı́nicas, Faculdade de
Medicina de Ribeirão Preto, São Paulo , Brazil

Received Sep 9, 2022; accepted Mar 14, 2023

doi:10.1007/s12350-023-03270-y

Background. Myocardial perfusion defect (MPD) is common in chronic Chagas car-

diomyopathy (CCC) and is associated with inflammation and development of left ventricular
systolic dysfunction. We tested the hypothesis that pentoxifylline (PTX) could reduce inflam-
mation and prevent the development of MPD in a model of CCC in hamsters.
Methods and results. We investigated with echocardiogram and rest myocardial perfusion
scintigraphy at baseline (6-months after T. cruzi infection/saline) and post-treatment (after
additional 2-months of PTX/saline administration), female Syrian hamsters assigned to 3
groups: T. cruzi-infected animals treated with PTX (CH 1 PTX) or saline (CH 1 SLN); and
uninfected control animals (CO). At the baseline, all groups showed similar left ventricular
ejection fraction (LVEF) and MPD areas. At post-treatment evaluation, there was a significant
increase of MPD in CH 1 SLN group (0.8 ± 1.6 to 9.4 ± 9.7%), but not in CH 1 PTX
(1.9 ± 3.0% to 2.7 ± 2.7%) that exhibited MPD area similar to CO (0.0 ± 0.0% to 0.0 ± 0.0%).
The LVEF decreased in both infected groups. Histological analysis showed a reduced
Supplementary Information The online version contains supple- Reprint requests: Marcus Vinı́cius Simões, PhD, Cardiology Division,
mentary material available at https://doi.org/10.1007/s12350-023- Internal Medicine Department, Hospital das Clı́nicas, Faculdade de
03270-y. Medicina de Ribeirão Preto, 3900 Bandeirantes Avenue, Ribeirão
The authors of this article have provided a PowerPoint file, available Preto, São Paulo 14048900, Brazil; msimoes@fmrp.usp.br
for download at SpringerLink, which summarises the contents of the J Nucl Cardiol 2023;30:2327–37.
paper and is free for re-use at meetings and presentations. Search for 1071-3581/$34.00
the article DOI on SpringerLink.com. Copyright Ó 2023 The Author(s) under exclusive licence to American
The authors have also provided an audio summary of the article, which Society of Nuclear Cardiology
is available to download as ESM, or to listen to via the JNC/ASNC
Podcast.

2327
2328 Tanaka et al Journal of Nuclear CardiologyÒ
Pentoxifylline reduces inflammation and prevents myocardial perfusion derangements in experimental November/December 2023

inflammatory infiltrate in CH 1 PTX group (395.7 ± 88.3 cell/mm2), as compared to CH 1

SLN (515.1 ± 133.0 cell/mm2), but larger than CO (193.0 ± 25.7 cell/mm2). The fibrosis and
TNF-a expression was higher in both infected groups.
Conclusions. The prolonged use of PTX is associated with positive effects, including pre-
vention of MPD development and reduction of inflammation in the chronic hamster model of
CCC. (J Nucl Cardiol 2023;30:2327–37.)
Key Words: Chronic Chagas cardiomyopathy Æ coronary microcirculation Æ inflammation Æ
ventricular dysfunction Æ hamsters

Abbreviations coronary microvascular dysfunction could have an

CCC Chronic Chagas’ cardiomyopathy
MPD Myocardial perfusion defect
PTX Pentoxifylline
PLB Placebo
LV Left ventricle
LVEDD Left ventricle end diastolic dimension
LVESD Left ventricle end systolic dimension
LVEF Left ventricular ejection function
SPECT Single-photon emission computerized
tomography
INTRODUCTION
More than 100 years ago, Trypanosoma cruzi (T.
cruzi) was identified as the causative agent of Chagas’
disease. This condition remains a neglected chronic
infectious disease with high health care burden.1 It is
estimated that 6.6 million people are infected globally,
mainly in Latin America, and emergent numbers in
Europe and the United States.1–3 The clinical course of
Chagas disease usually comprises an acute phase, which
is asymptomatic in most cases,4 and, once the acute
phase subsides, 20–30% of infected people may develop
a chronic Chagas’ cardiomyopathy (CCC) 20–30 years
after the infection.5
In addition to the main pathogenetic mechanisms
proposed to explain the development of chronic
myocarditis in CCC, clinical and experimental studies
suggest that myocardial perfusion disturbances (MPD),
caused by microvascular dysfunction, participate as a
lesion amplifier pathophysiological process.6,7 This is
closely linked to myocardial inflammation and is asso-
ciated with the development of platelet ‘‘plugs,’’
obstructive intimal proliferative lesions, arteriolar
spasm, and hypoperfusion,8,9 associated with increased
plasma cytokine levels in the CCC, primarily TNF- a
and IL-6.10,11
Clinical studies reported perfusion defects in 78%
of the CCC patients with normal epicardial coronary
arteries, associated with LV wall motion abnormalities12
and with the deterioration of LV systolic function over
time,13 strongly suggesting that MPD caused by
important role in the development of regional myocar-
dial dysfunction and damage in CCC.
More recently, studies in experimental models of
CCC in hamsters employing high-resolution scintigra-
phy images in vivo showed that MPD is topographically
correlated with higher intensity of the inflammatory
infiltrate.14
Therefore, it is plausible to suppose that pharma-
cological therapies addressing modulation of the
immune system and improvement of tissue perfusion
could positively impact myocardial perfusion in vivo
and attenuate the progression of ventricular dysfunction
in CCC.
We evaluated the effects of pentoxifylline, a phos-
phodiesterase inhibitor that modulates TNF-a synthesis
and increases tissue perfusion, on myocardial perfusion,
cardiac inflammation, and evolution of left ventricular
systolic dysfunction by using high-resolution in vivo
imaging methods in an experimental model of CCC in
Syrian hamsters.
METHODS
Experimental animals
Twelve-week-old female hamsters (Mesocricetus
auratus) (Anilab – Animais de Laboratório Criação e
Comércio Ltda, Paulı́nia/SP, Brasil) were used for the
study. They were maintained in a climatically controlled
environment based on a 12-h light/dark cycle with free
access to food and standard chow. All procedures and
protocols were approved by the Animal Research Ethics
Committee of the Institution (Protocol No. 025-2016).
Experimental protocol
The animals of the Chagas group were infected
intraperitoneally with 3.5 9 104 trypomastigote forms
of T. cruzi Y-strain. Control animals were inoculated
with the same volume of saline solution.
The detection of anti-T. cruzi antibodies confirmed
the chronic Chagas infection in the sera of the infected
hamsters with a Western blot assay with the TESA
fraction of secreted T. cruzi antigens employing anti-
Journal of Nuclear CardiologyÒ
Volume 30, Number 6;2327–37 Pentoxifylline reduces inflammation and prevents myocardial perfusion derangements in experimental
hamster IgG conjugated to horseradish peroxidase as the
secondary antibody as previously described.15
Six months after the T. cruzi infection, animals
were submitted to baseline imaging studies, including
high-resolution 99mTc-Sestamibi myocardial perfusion-
SPECT and echocardiography. We choose this time
window for the baseline evaluation based on previous
reports in hamster model of CCC, showing that at 6-
month after T. cruzi infection the animals present no
significant LV systolic dysfunction, which typically
develops at a later time window of 8-months after
infection.16
The animals were divided into 3 groups: 1. Con-
trol ? saline (CO, n = 9): non-infected animals treated
with saline. 2. Chagas ? saline (CH ? SLN, n = 12):
chronically T. cruzi-infected animals treated with saline;
Chagas ? pentoxifylline (CH ? PTX, n = 12): chroni-
cally T. cruzi-infected animals treated with
pentoxifylline.
The animals treated with pentoxifylline (Vascer,
União Quı́mica, Brazil) received intraperitoneal injec-
tions of 20 mg/kg daily for 60 days. This dose was
chosen based on previous studies investigating the effect
of pentoxifylline in rodent microcirculation.17 Placebo-
treated animals were injected intraperitoneally with the
same volume of saline solution.
In the post-treatment evaluation, animals underwent
the same imaging methods used at the baseline, followed
by euthanasia and the collection of heart tissue.
Echocardiographic assessment of left
ventricular structure and function
After sedation with ketamine and xylazine (100 and
10 mg/kg), 2D-Doppler-Echocardiography was recor-
ded using a dedicated ultrasound system (Vevo 2100,
VisualSonics, Toronto, ON, Canada) with a 30 MHz
high-frequency linear transducer. The conventional
echocardiography was acquired, as previously
described.18 The regional systolic function was deter-
mined by the average wall motion score index
(WMSi).19
The images were acquired by an echocardiographer
experienced in lab work with small animals who was
blind to the group to which the animals belonged for off-
line analysis at the end of the study.
High-resolution myocardial perfusion
SPECT
Rest myocardial perfusion images were acquired
with a validated high-resolution imaging system20,21
based on pinhole collimators coupled to a gamma
camera of clinical use (BrightView XCT; Philips
Tanaka et al 2329
Medical Systems Inc., Cleveland, OH). by using a
protocol previously described.22 Briefly, the animals
were anesthetized with isoflurane 3%, and 555 MBq of
99mTc-Sestamibi was injected into the sublingual vein.
After 90 min, animals were anesthetized with ketamine
(100 mg/Kg) and xylazine (10 mg/kg) intramuscularly
and positioned in the image acquisition system. Forty
projections equally spaced over 360 degrees were
acquired, 30 s/projection, using a 128 9 128 pixels
acquisition matrix. The usual pentoxifylline or placebo
administration was maintained in all experimental
groups until the radiotracer injection.
The images were processed to obtain the three-
dimensional radiopharmaceutical distribution in the
target organ using a dedicated reconstruction software.20
Images were reconstructed using a 128 9 128 matrix
(0.54 mm pixel size) and a three-dimensional ordered
subset expectation maximization algorithm (3D-OSEM).
No attenuation correction was applied. The semiquan-
titative analysis of myocardial perfusion was based on
the construction of polar maps of the relative distribu-
tion of the radiopharmaceutical in relation to the
maximum pixel count using Munich Heart softwareÒ
(Munich, Germany).20 Myocardial perfusion defects
were defined by those pixels exhibiting counts 2.5
standard deviations below the mean pixel uptake value
obtained in a normal hamster database. Defect areas
were expressed as percentage area in relation to the total
surface of the left ventricle. LV segmentation into a 13-
segment model (6 basal, 6 mid-ventricular, and 1 apical)
was used to determine the topographic correlation of the
perfusion defect areas with the histopathologic and
echocardiography studies.
Histopathology
After deep anesthesia, the chest was opened, and the
animals were sacrificed by exsanguination. The heart
was excised and washed in PBS solution, and basal
vessels and atria were discarded. For histopathological
analysis, short-axis sections were obtained at three
ventricular levels (basal, mid-ventricular, and apical).
Slices were scanned and analyzed with a digital pathol-
ogy system consisting of a digital scanner (Scanscope
CS System; Aperio Technologies Inc., Vista, CA, USA).
Quantitative analyses of inflammation were per-
formed by counting the number of mononuclear cell/
mm2 using Hematoxylin and Eosin staining. In addition,
the extent of interstitial and perivascular fibrosis was
quantified by Picrosirius-red staining, expressed as a
percentage area of fibrosis in relation to the total area of
the myocardial tissue. We used sampling areas ranging
from 1.5 to 2 mm2 in each myocardial segment for all
2330 Tanaka et al Journal of Nuclear CardiologyÒ
Pentoxifylline reduces inflammation and prevents myocardial perfusion derangements in experimental November/December 2023

histopathological analyses, maintaining topographic Statistical analysis

correlation with in vivo imaging exams.
Continuous variable data are reported as mean ± s-
tandard deviation, and nominal variables are reported as
mRNA expression absolute (n) and relative (%) frequency.
The Shapiro–Wilk test was used to determine the
A sample was taken from the right ventricle to
Gaussian distribution of variables.
analyze the gene expression of the tumor necrosis factor
Ordinary one-way ANOVA was used to compare
alfa (TNF-a), atrial natriuretic peptide (ANP), and
inflammation and Kruskal–Wallis simultaneously to
intercellular adhesion molecule (ICAM) by using RT-
compare fibrosis and gene expression. We used the
PCR technique, as previously described, as well as the
Mann–Whitney test to simultaneously compare two
sequences of all primers used. Briefly, the RNeasy
independent variables.
Fibrous tissue kit (Quiagen, USA) was used to isolate
A mixed-effect model ANOVA for repeated mea-
total RNA from right ventricle samples. RNase-free
sures was used to assess interaction (main effect)
DNase I treatment was applied to remove any DNA
between the experimental groups (between-subject
contamination. Subsequently, Super-script IITM reverse
effect) and time (within-subject effect) on LVEF,
transcriptase (Invitrogen, USA) was used to generate
LVEDD, and myocardial perfusion defect areas. Where
cDNA from 3 mg of total RNA. Real-time quantitative
a statistically significant interaction was found, simple
RT-PCR was performed using SYBR Green I PCR
effect tests were performed to evaluate differences
Master Mix (Applied Biosystems, USA) and 250 nM of
between pre- and post-treatment values into each
sense and anti-sense primers in the ABI Prism 7500
experimental group (Dunn’s multiple comparisons test
sequence detector (Applied Biosystems, USA). The
or Šı́dák’s multiple comparisons test). The significance
primers were designed using Primer Express software
level was set at p \ 0.05, two-tailed in all analyses.
version 3.0 (Table 1). After each PCR, an amplicon
Statistical analysis was performed using GraphPad
melting point curve was obtained, which yielded a single
Prism, version 9.1.2 (GraphPad Software, San Diego,
peak with the expected temperature provided by Primer
CA, USA).
Express software, thus confirming the specificity of the
PCR. The normalization of mRNA expression was
carried out using GAPDH mRNA expression. The RESULTS
amount of mRNA present in the right ventricles of
infected or infected treated animals compared to unin- LV structure and function
fected animals was calculated using the 2–DDCt
The echocardiogram results are presented in Table 2.
method.23,24
The echocardiography parameters were similar for all
groups at baseline evaluation.
In the post-treatment evaluation, both CH ? SLN
and CH ? PTX groups presented a significant reduction
of the LVEF, as compared to the baseline, Fig. 1. No

Table 1. Characteristics of the primers

Gene name GenBank accession number Sequence 50 ? 30 Amplicon (bp)

GAPDH DQ403055 ( ?)-CTGACATGCCGCCTGGAG 101
( -)-TCAGTGTAGCCCAGGATGCC
TNF-a AF315292 ( ?)-TCAAAATTCGAACGACAAGCC 102
( -)-TTGGCCAGGAGGGCATT
ANP D17313 ( ?)-GCCATATTGGAGCGAACCC 140
( -)-CATTCTGCTCACTCAGGGCC
ICAM DQ093373.1 ( ?)-ATTGTGGTGTACTCGTTCCCG 86
( -)-CAGTGCAGGGTGAGGTTCTT

GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TNF-a, tumor necrosis factor-a; ANP, atrial natriuretic peptide; ICAM,
intercellular adhesion molecule 1
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Volume 30, Number 6;2327–37 Pentoxifylline reduces inflammation and prevents myocardial perfusion derangements in experimental

difference was detected in the control group. We
observed an increase in the WMSi and LV dimensions
in both infected groups, but the differences did not reach
statistical significance (Table 2).
Myocardial perfusion
The results of the quantitative analysis of the rest
myocardial perfusion SPECT are summarized in
Table 2.
The mean value of MPD areas in the CH ? SLN
group ranged from 0.0 to 5.0% at baseline, and from 0.0
to 30.0% at post-treatment. In the CH ? PTX group, the
MPD areas ranged from 0.0 to 9.6% at baseline and from
0.0 to 7.0% at the post-treatment (Table 2). A significant
increase in the mean MPD areas was observed only in
the infected group treated with saline, Figs. 2 and 3.
Regarding the description of MPD at baseline, 4
animals in the CH ? SLN group (33%) had MPD,
involving 8 out of 52 analyzed segments (15.4%), with a
mean defect area of 2.5 ± 1.9% in these animals. In the
CH ? PTX group, 5 animals (41.7%) had areas with
MPD, involving 14 of the 65 segments analyzed
(21.5%), with a mean defect area of 4.7 ± 3.0% in
these animals. In the post-treatment analysis, we
observed in the CH ? SLN group, 9 animals (75%)
with MPD, involving 37 segments (71.2%), with a mean
defect area of 12.6 ± 9.3%. In the CH ? PTX group, 8
animals (66.7%), and 16 segments (24.6%), showed
perfusion defects, with a mean defect area of
4.1 ± 2.2%. Among the 17 animals of both Chagas
groups with post-treatment MPD, the most frequently
involved segments were: apex (88.2%), mid anterolat-
eral (76.5%), basal anterolateral (29.4%), and mid
posterolateral (29 0.4%).
Animals with MPD presented significantly lower
values of LVEF (38.5 ± 11.2%) when compared with
animals without MPD 48.4 ± 9.1% (p = 0.04), and a
trend to higher intensity of myocardial inflammation in
animals with MPD (540.4 ± 153.6 cell/mm2) vs. without
MPD (409.6 ± 130.3 cell/mm2), p = 0.09. In addition,
animals with MPD presented a significantly higher
ICAM (0.02 ± 0.01) expression when compared with
animals without MPD (0.01 ± 0.01), p = 0.02. There
was no significant difference between groups regarding
the extent of fibrosis. Moreover, an analysis based on
myocardial segments (n = 312) showed that segments
with MPD (n = 54) in comparison to those without
MPD (n = 258) presented a significantly higher number
of mononuclear cells (608 ± 299.9 cell/mm2 and
478.3 ± 201.1 cell/mm, respectively—p \ 0.0001), and
significantly higher WMSi (1.8 ± 0.9 and 1.2 ± 0.4,
respectively—p \ 0.0001).
Histopathological analysis
Infected animals exhibited intense focal inflamma-
tory infiltrate with mononuclear cells usually detected,

Table 2. Baseline and post-treatment results of echocardiographic and myocardial perfusion studies
for each experimental group

Variables Control (n = 9) CH 1 SLN (n = 12) CH 1 PTX (n = 12) p (Mixed-ANOVA)

LVEF (%)
Baseline 53.4 ± 5.6 47.0 ± 10.1 53.9 ± 6.5 0.03
Post-treatment 50.8 ± 4.1 40.0 ± 13.7 * 42.7 ± 9.0 *
LVEDD (mm)
Baseline 7.8 ± 0.5 7.8 ± 0.6 7.3 ± 0.5 0.3
Post-treatment 8.0 ± 0.4 8.2 ± 0.9 8.0 ± 0.7
PD (%)
Baseline 0.0 ± 0.0 0.8 ± 1.6 1.9 ± 3.0 0.004
Post-treatment 0.0 ± 0.0 9.4 ± 9.7 * 2.7 ± 2.7
WMSi
Baseline 1.0 ± 0.1 1.2 ± 0.3 1.1 ± 0.3 0.1
Post-treatment 1.0 ± 0.1 1.4 ± 0.5 1.3 ± 0.4

Data are reported as mean ± standard deviation

LVEF, left ventricular ejection fraction; LVEDD, left ventricular end-diastolic dimensions, PD, Perfusion defects; WMSi, Wall motion
score index; PTX, pentoxifylline; SLN, saline
*p \ 0.05 vs. baseline of the same experimental group,p \ 0.05 vs. post-treatment CH ? SLN, Šı́dák’s multiple comparisons
test
2332 Tanaka et al Journal of Nuclear CardiologyÒ
Pentoxifylline reduces inflammation and prevents myocardial perfusion derangements in experimental November/December 2023

80
*p=0.03

60
Control (n=9)
LVEF (%)

CH + SLN (n=12)
CH + PTX (n=12)
40 *
*

20
Baseline Post treatment

Figure 1. A Line graphs representing the mean and SD values of left ventricular ejection fraction
(LVEF) obtained for each experimental group at baseline and post-treatment with pentoxifylline or
saline. Mixed-ANOVA test.

25
*p=0.004 Control (n=9)
CH + SLN (n=12)
Perfusion defects (%)

20

15 CH + PTX (n=12)

10
*
5

-5
Baseline Post treatment

Figure 2. A Line graphs representing the values of myocardium perfusion defects areas obtained
for each experimental group at baseline and post-treatment with pentoxifylline or saline—Mixed-
ANOVA test.

and most animals exhibited mild multifocal myocarditis.
Preserved fibers without inflammatory changes were
detected in the control animals.
The number of mononuclear cells estimates the
intensity of the inflammatory infiltrate, and the extent of
fibrosis in each experimental group is reported in
Table 3. A larger number of mononuclear inflammatory
cells was detected in both Chagas groups compared to
controls. In addition, there was a significant difference
between the CH ? SLN and CH ? PTX groups, Fig. 4.
The extent of fibrosis was significantly larger in the
CH ? SLN and CH ? PTX groups compared to the
control group. We found no difference in fibrosis extent
between the 2 groups of infected animals treated with
saline or pentoxifylline, Fig. 4.
Analysis of mRNA expression
The summary of the analysis of mRNA expression
results is reported in Table 4. The mRNA expression of
TNF-a was significantly increased in infected animals,
with the post-test showing a significant difference
between Chagas ? SLN compared to the control group.
We found no difference between infected animals
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Volume 30, Number 6;2327–37 Pentoxifylline reduces inflammation and prevents myocardial perfusion derangements in experimental

Figure 3. Illustrative images of myocardial perfusion studies at baseline and post-treatment in an

infected animal treated with saline (A) or pentoxifylline (B). Representative sections of
tomographic images (SPECT) are shown. The images were obtained at the short axis of the left
ventricular cavity (basal, mid cavity, and apical) and on vertical and horizontal long axes, with the
resulting polar map and the computed defect area shown on the right side of the figure. In the
Chagas ? Saline animal, there was a significant increase in the MPD area post-treatment compared
to the baseline (1.2% to 30.3%). In the Chagas ? Pentoxifylline animal, the MPD area reduced
from 4.5% to 1.8%.

Table 3. Results of expression of histopathological analysis

p
Variables Control Chagas 1 SLN Chagas 1 PTX (Kruskal–Wallis)
Histopathology
Inflammation (cell/mm2) 193.0 ± 25.7 515.1 ± 133.0 * 395.7 ± 88.3 * \ 0.001
Fibrosis total (%) 0.56 ± 0.1 0.83 ± 0.2 * 0.88 ± 0.3 * 0.006

Data are reported as mean ± standard deviation

PTX, pentoxifylline; SLN, saline
*
p = 0.04 vs. control,p \ 0.03 vs CH ? PTX, Dunn’s multiple comparisons test
2334 Tanaka et al Journal of Nuclear CardiologyÒ
Pentoxifylline reduces inflammation and prevents myocardial perfusion derangements in experimental November/December 2023

Figure 4. Representative histological sections stained with HE (upper panels) and picrosirius
(lower panels) from each experimental group. The histological samples of infected animals show
focal points of a diffuse mononuclear inflammatory infiltrate and fibrosis. On the right side are the
bar graphs representing the quantitative analysis of inflammation and fibrosis results for each
experimental group. SLN, saline, PTX, pentoxifylline. The p-value of inflammation and fibrosis
refers to the Kruskal–Wallis test with Dunn’s post-test.

treated with saline or pentoxifylline. Also, ANP and
ICAM mRNA expression did not differ significantly
between the infected animals and controls.
DISCUSSION
The main results of our study showed that the
prolonged use of PTX in hamsters chronically infected
with T. cruzi prevented the development of rest MPD
and this effect was associated with a significant reduc-
tion of myocardial inflammation demonstrated by
histopathology.
These results, showing reduction of inflammation
associated with prevention of MPD, reinforces the
notion that MPD are intrinsically related to inflamma-
tion in this model of CCC. In fact, results of previous
experimental studies showed that myocardial areas with
rest perfusion disturbance corresponded to areas of
viable myocardial tissue, with no transmural scar, but
with increased inflammatory activity.14 Another previ-
ous study also showed amelioration of MPD with
prolonged use of dipyridamole providing additional
support to the notion that rest MPD in this CCC models
are closely related to microvascular dysfunction associ-
ated to myocardial inflammation.22
In the present study, the observed effect of PTX in
reducing both MPD and inflammation could be possibly
explained by 2 possible mechanisms, as follows. First, a
primary reduction of inflammation caused by immune
modulating effect of PTX mainly associated with a
potential reduction of TNF-a secretion, with consequent
amelioration of MPD owing to the attenuation of
microvascular dysfunction that is closely related to
inflammation in CCC.25,26

Table 4. Results of mRNA expression

Control Chagas 1 SLN Chagas 1 PTX p (Kruskal–Wallis)

mRNA expression*
TNF-a 0.86 ± 0.4 2.47 ± 1.7 2.42 ± 1.7 0.03
ANP 0.68 ± 0.5 2.70 ± 3.5 1.29 ± 1.5 0.4
ICAM 0.02 ± 0.01 0.02 ± 0.01 0.02 ± 0.01 0.6
*
2-dCt; p = 0.03 vs. control
Journal of Nuclear CardiologyÒ Tanaka et al 2335
Volume 30, Number 6;2327–37 Pentoxifylline reduces inflammation and prevents myocardial perfusion derangements in experimental

This mechanism would be in line with previous The lack of effect of pentoxifylline on left
studies in other disease scenarios, showing that PTX ventricular systolic dysfunction in CCC
treatment has a beneficial effect in improving cardiac
In our study, despite the positive effects on perfu-
function by reducing cardiac pro-inflammatory cytoki-
sion an inflammation, PTX did not prevent the
nes, TNF-a and IL6, that are also associated to
progression of LV systolic dysfunction. In concordance
endothelial dysfunction.27 These substances are known
with these results, the expression of ANP mRNA was
to induce overexpression of VCAM-1 and ICAM-1 in
increased in both T. cruzi infected animals in compar-
vascular endothelium, promoting the adhesion of neu-
ison to controls, although not reaching statistical
trophils which are essential for the pathogenesis of
significance, indicating similar degree of ventricular
vascular inflammatory diseases.26 The use of PTX has
wall stress and myocardial dysfunction in both CCC
also been shown to have strong protective effects on the
groups.
pulmonary endothelium in mice with pulmonary inflam-
Our results showing that PTX had a neutral effect
mation, preserving endothelial integrity, which may
on TNF-a production and left ventricular function, do
result from reduced TNF-a, and IL-6 release.25
not corroborate the results obtained by other studies that
Despite the preliminary expectancy, regarding an
used mice infected with T. cruzi in a shorter period after
effect of PTX in reducing myocardial TNF-a produc-
infection. Pereira et al. (2015), using mice infected with
tion, this was not confirmed by the present results. In our
the Colombian strain of T. cruzi, observed that after
study, both animal groups infected with T. cruzi pre-
30 days of PTX administration, there was a reversal of
sented increased levels of TNF-a mRNA expression,
electrocardiographic abnormalities, reduction of cardiac
confirming previous studies indicating that this cytokine
remodeling, and restoration of LVEF. Concurrent to
may be involved in the inflammation and progression of
these findings, there was a reduction in the number of
myocardial dysfunction in CCC.28,29 However, we
cells that express IFN-c, the expression of ICAM in
observed no reduction in TNF-a mRNA expression in
cardiac tissue, and the severity of myocarditis, with no
the group treated with PTX, making the contribution of
change in the parasite load.17 Subsequently, Vilar-
this mechanism for reducing inflammation less likely.
Pereira et al. (2016) also studied mice infected with
Despite this, our results agree with a previous study that
the Colombian strain of T. cruzi. They observed that
used a mouse model infected with T. cruzi and treated
PTX improved the balance of the immune response,
with pentoxifylline. In that study, no reduction in TNF-a
modulating the values of TNF-a and iNOS without
levels was observed, but only a reduction in TNF-a
interfering with the number of parasites.33
receptors was reported. In our study, we did not perform
This variability of response to PTX can be ascribed
analysis of TNF-a receptors. In addition, the results of
to several factors. First, it is known that the animal
the ICAM mRNA expression, that presented no differ-
model used can interfere with the pathogenicity of T.
ence in the group treated with PTX, further indicates that
cruzi, the development of the disease and response to
a mechanism involving reduction of inflammatory
therapeutic agents.34–37 It is essential to note that the
endothelial activation is not present.
host’s genetic profile can influence a series of reactions,
Second, an alternative explanation for the observed
especially concerning the immune response, which can
prevention of MPD development and reduction of
interfere from the onset of an acute symptomatic phase
inflammation associated to PTX, could be a predominant
until the development of the chronic phase,38–40 which
direct coronary microvascular vasodilation caused by
could explain the different interspecies responses
the phosphodiesterase inhibition effect of PTX that
observed in mice and hamsters as found here.
could contribute to reduce myocardial ischemia and
On the other hand, it is conceivable to suppose that
tissue injury, and with a secondary effect in attenuating
our results indicate that PTX has achieved an early
the intensity of myocardial inflammation. In fact,
effect of attenuation of inflammation and microvascular
previous studies in other models have shown that
dysfunction, but there was not sufficient time for these
inflammation is elicited by ischemia/reperfusion epi-
positive effects to impact LV systolic function, an effect
sodes and plays a relevant role in the myocardial injury
that could take longer to happen. This hypothesis may
process.30 In addition, PTX is also associated to other
gather support from previous studies in this same model
effects potentially contributing to amelioration of
of CCC, showing that MPD occur in the early stages of
myocardial perfusion, as reducing blood viscosity and
disease progression and may precede the development of
inhibition of platelet aggregation.31,32
LV systolic dysfunction.14,22 Similar findings were
reported in a longitudinal clinical study showing that
reversible MPD was topographically related to subse-
quent development of regional myocardial fibrosis and
2336 Tanaka et al
Pentoxifylline reduces inflammation and prevents myocardial perfusion derangements in experimental
deterioration of regional and global LV systolic function
in patients with CCC.13 These findings lend support to
the notion that, at early stages of disease evolution,
myocardial perfusion would be better than LVEF as a
non-invasive marker of disease progression and tissue
injury.41
We can also consider that the neutral effect of PTX
over LV systolic dysfunction could be attributed to the
initiation of PTX in an evolutive phase of CCC, at 6-
months after infection, in which tissue lesions have
already been set and causing the subsequent LV remod-
eling, regardless of the potential benefit associated to
PTX treatment over perfusion and inflammation. This
aspect could be further explored by future investigations
testing the effect of starting PTX in early time points
after T. cruzi infection.
NEW KNOWLEDGE GAINED
Coronary microvascular dysfunction associated
with a persistent diffuse low-grade myocardial inflam-
mation is a common finding in CCC. Our study aimed to
evaluate the effects of pentoxifylline on myocardial
perfusion, cardiac inflammation, and evolution of left
ventricular systolic dysfunction by using high-resolution
in vivo imaging methods in an experimental model of
CCC in Syrian hamsters. We present original results,
showing that prolonged use of pentoxifylline, an
immunomodulatory and vasodilator agent, is associated
with positive effects, including attenuation of inflam-
mation and prevention of the development of MPD in
this model. Our results indicate that MPD may be an
early marker of disease progression, and the assessment
of myocardial perfusion can be a helpful non-invasive
tool to monitor the progression of CCC.
CONCLUSIONS
Our results suggest that prolonged use of PTX is
associated with beneficial effects, including prevention
of MPD development and reduction of inflammation in
the evolution of the chronic model of CCC in hamsters.
Funding
This study was supported by a research grant from
Fundação de Apoio à Pesquisa do Estado de São Paulo
(FAPESP – Process: 2017/16450-6, 2016/25403-9 and 2019/
21250-1) and Fundação de Apoio ao Ensino Pesquisa e
Assisteˆncia do Hospital das Clı´nicas (FAEPA).
Disclosures
The authors declare that they have no conflict of interest.
Journal of Nuclear CardiologyÒ
November/December 2023
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