Acta Scientiae Veterinariae, 2014. 42: 1247.
RESEARCH ARTICLE
ISSN 1679-9216
Pub. 1247
TNF-α and IL-10 Modulate CK and CK-MB Activities in Naturally Infected Dogs by
Leishmania infantum
José Cláudio Carneiro de Freitas1, Adam Leal Lima1, Carla Tamára de Araújo Rodriguês1,
Gian Karlo Gama de Almeida1, Glauco Jonas Lemos Santos1, Bruno Mendes Roatt2,
Alexandre Barbosa Reis2 & Diana Célia Sousa Nunes-Pinheiro1
ABSTRACT
Background: Canine visceral leishmaniasis (CVL) is a multisystem inflammatory disease caused by the Leishmania genus
protozoa, characterized by increased production of pro-inflammatory mediators. The parasite presence has been reported
in various organs and tissues, such as bone marrow, lymph nodes, spleen, liver and skin, in addition the cardiac muscle.
Cardiac muscle lesions provide the substances release, among which we can mention the creatine kinase (CK), its MB
subunit (CK-MB) and troponin. The changes that cause these injuries depend not only of the parasite presence, but also
the increased concentrations of proinflammatory cytokines. Although most studies focus on the proinflammatory cytokines
action, regulatory cytokines of the inflammatory process has gained evidence in the immune response to the Leishmania
infantum and can be directly associated with the cardiac damage pathogenesis. Therefore, this study aimed to evaluate the
TNF-α and IL-10 serum levels in naturally infected dogs by L. infantum and relates them to cardiac biomarkers, in the
different clinical forms of disease.
Materials, Methods & Results: A total of 30 adult dogs were used, being 10 negative (ND) and 20 positive for CVL.
All seropositive animals were subjected to clinical examination, observing the presence of characteristic clinical signs of
disease, being divided into two groups: asymptomatic (AD, n = 10) and symptomatic (SD, n = 10) dogs. Blood samples
from all animals were collected to obtain serum for subsequent measurement of TNF-α, IL-10, CK and CK-MB. Data
were analyzed by Kruskal-Wallys test, followed by Dunn’s test. The correlation and influence of TNF-α, IL-10 and IL-10/
TNF-α on CK, CK-MB and CK-MB/CK, were determined by Spearman correlation test and linear regression. To evaluate
the relationship between the clinical signs onset and the TNF-α, IL-10, IL-10/TNF-α, CK, CK-MB and CK-MB/CK serum
levels, the unpaired t-test was used. TNF-α and IL-10, in the different groups, showed no significant differences. Already
IL-10/TNF-α ratio was significantly increased in AD, when compared to ND. CK was significantly increased in infected
animals, when compared to ND. Already the CK-MB was significantly increased in SD, when compared to ND. CK-MB/
CK ratio, in the different groups, showed no significant differences. TNF-α and IL-10 showed a moderate correlation and
weak negative and positive influence on CK and CK-MB, in AD and SD. The IL-10/TNF-α ratio presented a weak correlation and moderate negative influence in AD, and moderate correlation and strong positive influence, in SD, on CK and
CK-MB. It was also observed weak and moderate correlations, besides the weak and strong positive influences, of the
IL-10/TNF-α ratio on CK-MB/CK ratio, in AD and SD dogs.
Discussion: The elevation of CK and CK-MB serum levels, in asymptomatic and symptomatic animals, is directly related
to the impairment of skeletal muscles, due to the increased production of proinflammatory cytokines, since the analysis of
a cardiac marker more specific, such as troponin I, excluded the possibility of heart damage. Furthermore, our results suggest that the L. infantum infection can trigger cardiovascular disorders, regardless of their relationship with the increased
production of TNF-α. However, CK and CK-MB, separately evaluated, were not presented as good markers of cardiac
damage in naturally infected dogs by L. infantum.
Keywords: canine visceral leishmaniasis, cardiac biomarkers, TNF-α, IL-10.
Received: 2 August 2014
Accepted: 28 November 2014
Published: 24 December 2014
Laboratório de Imunologia e Bioquímica Animal, Programa de Pós-graduação em Ciências Veterinárias, Faculdade de Veterinária, Universidade
Estadual do Ceará (UECE), Fortaleza, CE, Brazil. 2Laboratório de Imunopatologia, Núcleo de Pesquisa em Ciências Biológicas (NUPEB), Instituto de
Ciências Exatas e Biológicas (ICEB II), Universidade Federal de Ouro Preto (UFOP), Ouro Preto, MG, Brazil. CORRESPONDENCE: J.C.C. Freitas
[joseclaudiocarneiro@yahoo.com.br - Fax: +55 (85) 3101-9860]. Avenida Dr Silas Munguba n. 1700, Campus do Itaperi, Serrinha. CEP 60740-903
Fortaleza, CE, Brazil.
1
1
J.C.C. Freitas, A.L. Lima, C.T.A. Rodriguês, et al. 2014. TNF-α and IL-10 Modulate CK and CK-MB Activities in Naturally Infected
Dogs by Leishmania infantum.
Acta Scientiae Veterinariae. 42: 1247.
INTRODUCTION
heartworm excluding, a chromatographic immunoassay2 (Anigen Rapid Heartworm Ag Test) was used for
the qualitative detection of Dirofilaria immitis antigen
according to manufacturer’s instructions.
Thirty animals were divided into three groups
(n = 10), according to the serology and the clinical signs
onset: negative dogs (ND), composed of serologically
negative dogs for CVL; asymptomatic dogs (AD), consisting of seropositive dogs for CVL, and who had no clinical
signs characteristic of the disease and symptomatic dogs
(SD), composed of seropositive dogs for CVL, and had
at least three characteristic clinical signs of disease. AD
and SD were from the CCZ, as those belonging to ND
were from private kennels. All clustered animals were
serologically negative for canine heartworm disease.
Animals were immobilized and blood samples
were collected with sterile syringe by jugular venipuncture, being distributed in tubes without anticoagulant.
Furthermore, the blood samples were centrifuged at
4000 g, for 10 min, to obtain the serum which were
stored at frozen (-20ºC) until use.
Canine visceral leishmaniasis (CVL) is a multisystem inflammatory disease caused by the Leishmania
genus protozoa, characterized by increased production
of pro-inflammatory mediators, generated by excessive
immune response and culminating in tissue damages
[18]. The presence of the parasite has been reported in
various organs and tissues, such as bone marrow [15],
lymph nodes [2], spleen [33], liver [16] and skin [28],
in addition the cardiac muscle [25,27].
Cardiac muscle lesions provide substances
releasing, among which we can mention the kinases,
creatine kinase (CK) and its MB subunit (CK-MB),
and troponin I [14,32]. It has been reported that the
changes causing these injuries depend not only of
the parasite presence, but also the proinflammatory
cytokines increased concentrations, such as TNF-α,
INF-γ, IL-6 and IL-12 [3,39]. These cytokines can
directly contribute to the cardiac injury progression
in the muscle fiber, as well as the CVL clinical manifestations onset, particularly those related to reduced
muscle mass [17,33].
Although most studies focus on the proinflammatory cytokines action, regulatory cytokines of
the inflammatory process, such as IL-10, has gained
evidence in the immune response to the L. infantum
and can be directly associated with the cardiac damage
pathogenesis [29,37]. Furthermore, studies have shown
that IL-10/TNF-α ratio is more accurate in detecting
heart function loss [19,23].
Based on this, the aim of this study was to
evaluate the TNF-α and IL-10 serum levels in naturally
infected dogs by L. infantum, and relate them to the
cardiac enzyme biomarkers, CK and CK-MB, in different clinical forms of the disease.
Semi-quantitative analysis of troponin I serum levels
Troponin I serum levels were determined by
immunochromatographic test3 (Troponina I Test K085)
according to the manufacturer’s instructions. The animals considered positive have troponin I serum levels
≥ 0.5 ng/mL.
CK and CK-MB serum levels determination
CK and CK-MB activities were determined
by using a commercial kit4 (CK and CK-MB Test), at
automatic biochemical. For the measurement of CKMB isoenzyme, previously sera underwent to heating
in water bath at 45°C, for 20 min, before the samples
are taken to the dosing automatic biochemical. The
values obtained were compared with reference values
for the canine species, providing essential data to
characterize the animals.
MATERIALS AND METHODS
Animals
TNF-α and IL-10 serum levels determination
Dogs of different ages, irrespective weight,
breed and sex, from the Zoonosis Control Center of
Fortaleza city (CCZ) and private kennels were used.
All animals underwent a complete clinical examination, performed by appropriately trained professionals,
and serological screening test for chromatographic
immunoassay1 and confirmatory enzyme linked immunosorbent assay (ELISA)1 for CVL were performed
according to manufacturer’s instructions. For canine
Cytokines levels were determined by enzymelinked immunosorbent assay (ELISA), according to the
manufacturer’s instructions. DuoSet ELISA5 (Immunoassay - R&D Systems) was used for analysis of anti-canine
TNF-α and IL-10. Minimum sensitivity was 63 ρg/mL
for TNF-α. All experiments were performed using 96-well
plates. The reading was performed using the microplate
automatic reader, at a wavelength of 450 nm.
2
J.C.C. Freitas, A.L. Lima, C.T.A. Rodriguês, et al. 2014. TNF-α and IL-10 Modulate CK and CK-MB Activities in Naturally Infected
Dogs by Leishmania infantum.
Acta Scientiae Veterinariae. 42: 1247.
different clinical forms, were determined by Spearman
correlation test and linear regression, respectively. To
evaluate the relationship between the onset of clinical
signs and the TNF-α, IL-10, IL-10/TNF-α ratio, CK,
CK-MB and CK-MB/CK ratio serum levels (P value) the
unpaired t-test was used. All analyzes were performed
using the statistical program GraphPad Prism 5.0, with
a significance level of 5%.
Statistical Analysis
TNF-α, IL-10, IL-10/TNF-α ratio, CK, CK-MB
and CK-MB/CK ratio serum levels were expressed as
mean and standard deviation and analyzed by KruskalWallys test, followed by Dunn’s test, for comparison
of three groups (ND, AD and SD). The correlation and
influence of TNF-α, IL-10 and IL-10/TNF-α ratio serum
levels on CK, CK-MB and CK-MB/CK ratio, in the
Table 1. Mean of TNF-α, IL-10, IL-10/TNF-α ratio, CK, CK-MB and CK-MB/CK ratio serum levels in naturally infected
dogs by L. infantum and their respective reference values for canine species.
Parameter
TNF-α (ρg/mL)
IL-10 (ρg/mL)
IL-10/TNF-α Ratio
CK (U/L x100)
CK-MB (U/L x100)
CK-MB/CK Ratio
ND
39.36±3.14
17.57±0.86
0.46±0.17
0.82±0.04
0.55±0.12
0.68±0.04
AD
35.43±2.15
25.62±1.02
0.71±0.30
1.58±0.81
1.36±0.22
0.61±0.13
SD
37.25±3.04
24.93±1.78
0.64±0.15
1.86±0.06
1.38±0.29
0.59±0.11
Reference*
a
≤10.0
b
≤10.0
-------------c
0.24 - 1.70
c
0.11 - 0.39
c
0.19 - 0.69
* aAccording to Resende et al. [34]. bAccording to Boggiatto et al. [8]. cAccording to Lopes et al. [24].
significantly increased in asymptomatic animals,
when compared to the negative animals (P < 0.05).
SD showed no significant difference, when compared
to ND and AD (P < 0.05) (Figure 1). The correlation
and influence of TNF-α, IL-10 and IL-10/TNF-α ratio
serum levels on CK, CK-MB and CK-MB/CK ratio,
in AD and SD groups, were expressed in Figures 2,
3, 4 and 5.
TNF-α serum levels showed a moderate correlation (ρ = -0.55 and ρ = 0.38) and weak negative
(y = 4.79 - 0.09x) and positive influence (y = 0.16 +
0.04x) on CK serum levels, in AD and SD, respectively
(Figure 2). The same way occurs with respect to CKMB serum levels, in AD (ρ = -0.2 and y = 2.05 - 0.02x)
and SD (ρ = 0.5 and y = 0.84 + 0.04x), respectively
(Figure 3).
IL-10 serum levels also showed moderate correlation (ρ = -0.2, ρ = 0.5, ρ = -0.05 and ρ = 0.85) and
weak negative influence (y = 2.05 - 0.02x and y = 1.82
- 0.02x), in AD, and positive (y = 0.84 + 0.04x and y
= -0.02 + 0.06x), in SD, both as compared to CK and
CK-MB, respectively (Figures 2 and 3).
IL-10/TNF-α ratio presented a weak correlation
(ρ = -0.4 and ρ = 0.05), and moderate negative influence (y
= 2.05 - 0.67x and y = 1.68 - 0.44x), in AD, and moderate
correlation (ρ = 0.54 and ρ = 0.78) and strong positive
influence (y = -0.2 + 3.35x and y = -1.77 + 4.91x), in SD,
on CK and CK-MB serum levels, respectively (Figure 4).
RESULTS
The mean values of TNF-α (ρg/mL), IL-10
(ρg/mL) IL-10/TNF-α ratio, CK (U/L x100), CK-MB
(U/L x100) and CK-MB/CK ratio serum levels, in all
groups (ND, AD and SD), were shown in Table 1 and
Figure 1. The relationship between the clinical signs
onset and serum levels of TNF-α, IL-10, IL-10/TNF-α
ratio, CK, CK-MB and CK-MB/CK ratio (P value)
were shown in Table 2.
CK serum levels were significantly increased
in infected animals (AD and SD), when compared to
negative animals (ND). Already the CK-MB serum
levels were significantly increased in symptomatic animals (SD), when compared to negative animals (ND).
However, the asymptomatic animals (AD) showed
no significant differences, in CK-MB serum levels,
when compared to animals belonging to ND and SD.
CK-MB/CK ratio, in the different groups, showed no
significant differences (P < 0.05) [Figure 1]. CK, CKMB and CK-MB/CK ratio serum levels did not present
relation with the characteristic clinical signs onset
(P < 0.05) [Table 2]. Additionally, all animals (ND,
AD and SD) were negative to troponin I test, ie, with
serum levels <0.5 ng/mL.
Evaluation of TNF-α and IL-10 serum levels,
in the different groups, showed no significant differences (P < 0.05). Already IL-10/TNF-α ratio was
3
J.C.C. Freitas, A.L. Lima, C.T.A. Rodriguês, et al. 2014. TNF-α and IL-10 Modulate CK and CK-MB Activities in Naturally Infected
Dogs by Leishmania infantum.
Acta Scientiae Veterinariae. 42: 1247.
It was also observed weak and moderate correlations (ρ =
0.08 and ρ = 0.59), besides the weak and strong positive
influences, of the IL-10/TNF-α ratio on CK-MB/CK ratio
(y = 0.78 + 0.03x and y = -0.21 + 1.58x), in AD and SD,
respectively (Figure 5). Furthermore, it was observed that
the TNF-α, IL-10, IL-10/TNF-α ratio serum levels did
not present relation with the characteristic clinical signs
onset (P < 0.05) [Table 2].
Table 2. P value of TNF-α, IL-10, IL-10/TNF-α ratio, CK, CK-MB and CK-MB/CK ratio
serum levels in naturally infected dogs by Leishmania infantum. Where P compares the
differences in serum levels from asymptomatic (AD) and symptomatic (SD) dogs.
Parameter (AD x SD)
P value
TNF-α
0.55
IL-10
0.90
IL-10/TNF-α Ratio
0.50
CK
0.51
CK-MB
0.97
CK-MB/CK Ratio
0.99
Figure 1. TNF-α, IL-10, IL-10/TNF-α ratio, CK, CK-MB and CK-MB/CK ratio in the different clinical forms of
naturally infected dogs by Leishmania infantum. Different letters represent differences at a significance level of 5%.
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J.C.C. Freitas, A.L. Lima, C.T.A. Rodriguês, et al. 2014. TNF-α and IL-10 Modulate CK and CK-MB Activities in Naturally Infected
Dogs by Leishmania infantum.
Acta Scientiae Veterinariae. 42: 1247.
Figure 2. Correlation and influence of TNF-α and IL-10 on CK serum levels, in the different clinical forms (AD and SD), in
naturally infected dogs by Leishmania infantum.
Figure 3. Correlation and influence of TNF-α and IL-10 on CK-MB serum levels, in the different clinical forms (AD and
SD), in naturally infected dogs by Leishmania infantum.
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J.C.C. Freitas, A.L. Lima, C.T.A. Rodriguês, et al. 2014. TNF-α and IL-10 Modulate CK and CK-MB Activities in Naturally Infected
Dogs by Leishmania infantum.
Acta Scientiae Veterinariae. 42: 1247.
Figure 4. Correlation and influence of IL-10/TNF-α ratio on CK and CK-MB serum levels, in the different clinical forms (AD and SD), in naturally infected dogs by Leishmania infantum.
Figure 5. Correlation and influence of IL-10/TNF-α ratio on CK-MB/CK ratio, in the different clinical forms (AD and
SD), in naturally infected dogs by Leishmania infantum.
disease development [2,9,20], being associated with
the parasites persistence in the lesions [1,7].
In the present study, TNF-α and IL-10 serum levels (Figure 1), in naturally infected dogs by L. infantum,
both symptomatic (SD) and asymptomatic (AD), showed
no significant differences when compared to non-infected
animals (ND). Already when evaluating IL-10/TNF-α
ratio, significant increase was observed in AD, when
compared to SD and to ND (Figure 1), showing the
IL-10 predominance, in these animals, in relation to
TNF-α. However, it has been reported that elevated IL-
DISCUSSION
TNF-α is an important cytokine that is related to proinflammatory activity; on the other hand,
IL-10 is a cytokine associated to immunoregulatory
activity. TNF-α has been associated to the infections
control caused by intracellular parasites, through the
Th1-mediated response activation [2,6] while IL-10
modulate the Th1-mediated response and it prevents
host damages caused by exacerbated inflammatory
responses [4,29]. In CVL, IL-10 inhibits the macrophages and dendritic cells response, facilitating the
6
J.C.C. Freitas, A.L. Lima, C.T.A. Rodriguês, et al. 2014. TNF-α and IL-10 Modulate CK and CK-MB Activities in Naturally Infected
Dogs by Leishmania infantum.
Acta Scientiae Veterinariae. 42: 1247.
10 and TNF-α serum levels, in naturally infected dogs by
L. infantum, are associated, respectively, to the onset or
not of disease clinical signs [6]. Here, we can also observe
that there was no relationship of IL-10 and TNF-α serum
levels with the clinical signs onset of the disease (Table 2).
It has been demonstrated that the affected
animals by visceral leishmaniasis presented cardiac
lesions [25,27]. To evaluate the parasite influence on
biomarkers related to heart diseases, the confirmation
of doubtful cases, to establish prognosis and deciding
on a specific treatment [26,30], it is necessary to evaluate the clinical signs onset. In this work it was used the
measurement of troponin I, creatine kinase (CK), and
its MB subunit (CK-MB) serum levels and its correlation with IL-10 and TNF-α serum levels.
CK is identified as a highly sensitive and
specific biomarker of muscle injury, which may be
expressed in skeletal and cardiac muscle tissue [13].
As CK is an extravasation enzyme, in cases of reversible muscle damage or necrosis, may be an increase in
their serum levels in animals [5,12]. In this study, CK
serum levels were significantly increased in AD and
SD, regardless of their clinical presentation, compared
to ND (Figure 2). However, the mean values, of ND
and AD, were within of the reference values for the
canine species and increased, in SD, when compared to
reference values (0.24 - 1.70 U/L x100) [24] (Table 1).
Significant increase in CK-MB serum levels in
SD was observed in the present study, when compared
to ND (Figure 2). A similar result was described in dogs
with and myocardial damage secondary to parvovirus,
heartworm, endocarditis, hypertrophic cardiomyopathy
and CVL [10,27,31,36], being CK-MB described as a
sensitive and specific cardiac marker, can be used for
detection of myocardial injury in these diseases. When
compared to the reference values of the canine species
(0.11 - 0.39 U/L x100) [24], the CK-MB serum levels,
in this study, were significantly increased (Table 1).
CK-MB serum levels are pointed as biomarker
of cardiac and muscular injury [5,40]. However, it
has been reported that elevated CK-MB serum levels
cannot be directly associated with myocardial impairment, since it can also be found in other tissues, even
in smaller amount, thus lowering their specificity [13].
Furthermore, macromolecules can be formed (macro
CK 1 and 2), altering the results of the measurement,
inducing false-positive data [22]. To eliminate this
interference in our study the samples were heated.
The assessment of troponin I has high sensitivity and specificity in the detection of cardiac damage in dogs [35]. Troponin I blood release occurs by
dissociation of the contractile apparatus and relate to
irreversible myocardial cell injury [32]. Here, it can be
related that none of the dogs, both symptomatic and
asymptomatic, presented troponin I serum levels above
0.5 ng/mL, suggesting the absence of heart muscle
damage. Similar values were obtained in a study performed by Santos et al. [35] in animals demonstrably
without myocardial damage. These data could indicate
that the increase of CK and CK-MB serum levels is
directly associated with impairment of the skeletal
muscle integrity [5].
The muscular and cardiac changes can be
directly related to the action of inflammatory mediators, such as TNF-α, IL-1β and IL-6, which may act
directly on the functionality and structure of cardiac
and skeletal muscle [17,23,39]. The increased production of proinflammatory cytokines, in CVL, has been
described [11,21,28].
In our work, TNF-α and IL-10 serum levels
did not differ significantly among the groups (ND, AD
and SD) [Figure 1]. However, both cytokines showed
increased serum concentrations, when compared to the
reference values (< 10 ρg/mL), according to Resende et
al. [34], for TNF-α, and Boggiatto et al. [8], for IL-10
(Table 1). However, it have been reported significantly
increased TNF-α serum levels in AD when compared to
SD [28]. In this study, TNF-α showed a slight negative
influence on CK (Figure 3) and CK-MB (Figure 4) in
AD. However, slight positive influence on the same
biomarkers, in symptomatic dogs was observed. IL-10
behaved similarly to TNF-α, regardless of the animal
clinical status, in relation to the cardiac biomarkers
(CK and CK-MB).
Our findings demonstrate the predominance
of IL-10 in AD, and TNF-α in SD. These results may
be associated to increase of others pro-inflammatory
cytokines production, which can potentiate the TNF-α
action and promote degenerative effect on cardiac and
skeletal muscle. Similar results were observed for in
humans and rats, where also there was verified the
remodeling effect of heart muscles associated with the
action of TNF-α and consequent production of metalloproteinases [37,38].
IL-10/TNF-α ratio evaluation on CK and CKMB serum levels (Figure 5) corroborate with greater
7
J.C.C. Freitas, A.L. Lima, C.T.A. Rodriguês, et al. 2014. TNF-α and IL-10 Modulate CK and CK-MB Activities in Naturally Infected
Dogs by Leishmania infantum.
Acta Scientiae Veterinariae. 42: 1247.
emphasis on what has been described previously,
where it was observed a slight negative influence, in
AD, highlighting the predominance of the IL-10 action, and a strong positive influence in SD, suggesting
the participation of other pro-inflammatory cytokines,
with increased production in CVL, in addition to the
effects of TNF-α. Furthermore, we may report that, in
the present study, none of the biomarkers assessed has
influenced the characteristic clinical signs onset (Table
2). This reinforces the hypothesis of the involvement
of other cytokines that can potentiate this effect.
Therefore, in summary, the increase of CK
and CK-MB serum levels in AD and SD is directly
related to the impairment of skeletal muscles, due to
the elevated production of proinflammatory cytokines,
since the analysis of a cardiac marker more specific,
such as troponin I, excludes the possibility of heart
damage.
Furthermore, our results suggest that the
L. infantum infection can trigger cardiovascular disorders, regardless of their relationship with the increased
production of TNF-α. However, CK and CK-MB,
individually evaluated, were not presented as good
markers of cardiac damage in naturally infected dogs
by L. infantum. More studies are needed for the understanding of the molecular mechanisms that modulate
the production, as well as, the participation of other
cytokines that may be involved in the process.
MANUFACTURERS
Instituto de Tecnologia em Imunobiológicos (Bio-Manguinhos). Rio
1
de Janeiro, RJ, Brazil.
Bioeasy Diagnóstica Ltda. São Paulo, SP, Brazil.
2
Bioclin Inc. Brussels, Belgium.
3
Wiener Lab Group. Rosario, Argentina.
4
R&D Systems. Minneapolis, MN, USA.
5
Funding. This work was financial supported by Coordenação de
Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) and
Fundação Cearense de Apoio ao Desenvolvimento Científico e
Tecnológico (FUNCAP).
Ethical approval. The experimental procedures were performed in accordance with the ethical principles of animal
experimentation, adopted by the Brazilian College of Animal
Experimentation (COBEA) and was approved by the Ethics
Committee for Animal Use of the State University of Ceará,
Protocol No. 12783812-0.
Declaration of interest. The authors report no conflicts of
interest. The authors alone are responsible for the content and
writing of the paper.
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