Different diseases or tissue injuries can produce changes in the central or peripheral nervous system, resulting in persistent pain. The development of therapeutics to treat chronic pain has been particularly challenging. Peripheral hypersensitivity induced by inflammation or nerve injury may involve a number of complex chemical mediators that may directly or indirectly interact with sensory nerves.1 Clinical trials have identified some effective drugs to treat chronic pain,2 such as tricyclic antidepressants, opioids, topical sodium channel-blockers, and drugs with antiepileptic properties. Interestingly, none of these drugs was developed to treat chronic pain.3 However, the available therapies only partially reverse pain and lead to the development of significant side effects, which culminate in treatment disruption.4
5-Carboxy-2,3-dihydro-2-(1′,5′-dimethyl-1′-hydroxy-4′-hexenyl)-7-(3”-methyl-2”-butenyl) benzofuran, also known as myrsinoic acid B (MAB, Fig. 1), is a prenylated benzoic acid found in the vegetal kingdom, mainly in plants of the Rapanea genus.5,6,7,8,9 Studies have demonstrated that MAB, administered topically, has significant anti-inflammatory effects in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema in mice.8 Moreover, Hess et al.9 have demonstrated that MAB was able to inhibit nociception induced by acetic acid, acting through α-adrenergic, serotonergic, nitric oxide, and cholinergic systems. The present study aimed to investigate the antihypersensitivity effect of MAB using different experimental models of inflammatory and neuropathic pain in mice, and efforts were made to determine the possible mechanism of action.
General Procedures for the Isolation of Myrsinoic Acid B
For the extraction and isolation of MAB, the peels of Rapanea ferruginea (600 g) were dried in a 40°C oven for up to 2 days, weighed and macerated with CHCl3 for 7 days at room temperature. The extract was concentrated under reduced pressure. CHCl3 extract (60 g) was chromatographed using silica gel columns with hexane/ethyl acetate. The fraction eluted with 40% hexane/ethyl acetate resulted in impure MAB. This fraction (8.5 g) was rechromatographed using silica gel columns with hexane/ethyl acetate to obtain pure MAB (4.3 g).
Female Swiss mice (25 to 30 g), obtained from the Universidade Do Vale Do Itajaí (UNIVALI, Itajaí, Brazil), were used in this study. Animals were housed under conditions of optimum light, temperature, and humidity (12-h light-dark cycle, 22 ± 1°C, 60% to 80% humidity), with food and water provided ad libitum. All procedures used in the present study followed the “Principles of Laboratory Animal Care” from National Institutes of Health publication No. 85 to 23 and were approved by the Animal Ethics Committee of UNIVALI (Protocol numbers 428/2007 UNIVALI). All efforts were made to minimize animal suffering and reduce the number of animals used.
Mechanical Hypersensitivity Induced by Carrageenan
Female mice received an intradermal (i.d.) injection of 50 μL of carrageenan (300 μg/paw) in the surface of the right hindpaw.10 To assess the acute effect of drug treatment, mice were pretreated orally with MAB (8.37, 27.93, or 83.79 μmol/kg) or indomethacin (13.9 μmol/kg; used as positive control) 1 hour before the carrageenan injection. Control animals received vehicle (10 mL/kg) by the same route of administration. Mechanical hypersensitivity of all groups was assessed up to 72 hours after carrageenan administration, as described below. To evaluate the site of action (central or peripheral) of MAB, the animals were treated through intracerebroventricular (i.c.v. 0.08, 0.27, or 0.83 nmol/site, 10 min before), intrathecal (i.t.; 8.3, 27.83, or 270 pmol/site, 10 min before), IV (0.83, 2.79, or 8.37 μmol/kg, 10 min before) or i.d. (8.37, 27.93, or 83.79 nmol/paw, coadministered with carrageenan) injections. Control animals received a similar volume of vehicle used for MAB dilution via the same injection sites: i.c.v. (5 μL/site), i.t. (5 μL/site), i.d. (50 μL/paw), or IV (10 mL/kg).
Mechanical Hypersensitivity Induced by Complete Freund Adjuvant
To produce a persistent inflammatory response, mice received an i.d. injection of 20 μL of complete Freund adjuvant (CFA) (20 μL/paw; 1 mg/mL heat-killed and dried Mycobacterium tuberculosis; each mL of vehicle contained 0.85 mL paraffin oil plus 0.15 mL mannide monooleate) in the plantar surface of the right hindpaw.10 To investigate the preventive effect, MAB (8.37, 27.93, or 83.79 μmol/kg), gabapentin (0.4 mmol/kg), or vehicle (10 mL/kg) was administered orally 1 hour before CFA injection, and mechanical hypersensitivity was evaluated at different time points (1 to 48 hours) using a 0.6 g von Frey filament. To evaluate the therapeutic effect of MAB, mice received CFA injections, and after 24 hours they were treated orally with MAB (8.37, 27.93, or 83.79 μmol/kg), gabapentin (0.4 mmol/kg), or vehicle (10 mL/kg) twice a day (every 12 hours) for a period of 5 days. Mechanical hypersensitivity was evaluated 6 hours after the first daily administration.
Mechanical Hypersensitivity Induced by Partial Ligation of the Sciatic Nerve)
Animals were anesthetized with 7% chloral hydrate (6 mL/kg; intraperitoneal), and partial ligation of the sciatic nerve (PLSN) was performed by tying 1/3 to 1/2 of the dorsal portion of the sciatic nerve. In sham-operated control groups, the sciatic nerve was exposed without ligation.10,11 Operated animals were administered MAB (8.37, 27.93, or 83.79 μmol/kg, PO), gabapentin (0.4 mmol/kg, PO), or vehicle (10 mL/kg, PO) 4 days after surgery (after a period of recovery), twice a day (every 12 hours) for 5 days.
Behavioral Pain-Related Variables
Mice were individually placed in clear Plexiglass boxes (9 × 7 × 11 cm) on elevated wire mesh platforms to allow access to the ventral surface of the hindpaws. The withdrawal response frequency was measured after 10 applications of a von Frey hair (VFH; Stoelting, Chicago, IL). Previous studies in our laboratory have indicated that the 0.6 g VFH produces a mean withdrawal frequency of about 15%, which was considered an adequate value for the measurement of mechanical hypersensitivity.10 Therefore, only the 0.6 g VFH was used in these experiments.
Thermal Hypersensitivity: Hot Stimulus
Thermal hypersensitivity was measured using the paw-withdrawal latency method, as described by Hargreaves et al.,12 with minor modifications. Mice were placed in clear plastic chambers (7 × 9 × 11 cm) on an elevated surface and allowed to acclimatize to their environment for 1 hour before testing. The heat stimulus was directed to the plantar surface of each hindpaw. The infrared intensity was adjusted to obtain a basal paw-withdrawal latency of ∼8 s. An automatic 20-second cutoff was used to prevent tissue damage.
Thermal Hypersensitivity: Cold Stimulus
To measure cold hypersensitivity, mice were individually placed into glass cylinders 20 cm in diameter, and 20 μL of acetone was applied to the right paw of PLSN-treated animals and sham-operated controls. The amount of time spent licking and/or biting the paw, which was considered to be indicative of hypersensitivity, was timed with a chronometer for 5 minutes.13
Neutrophil recruitment to the mouse paw was indirectly assessed by measuring tissue myeloperoxidase (MPO) activity, as described previously.14 For this purpose, animals were treated with MAB (8.37, 27.93, or 83.79 μmol/kg, PO), and after 1 hour, they received a 50-μL i.d. injection of carrageenan (300 μg/paw) into the right hindpaw. Another group of mice received saline into the right hindpaws and were used as a control. Animals were killed 6 hours after the application of carrageenan. The subcutaneous tissue of the paws was removed, homogenized at 5% (w/v) in EDTA/NaCl buffer (pH 4.7), and centrifuged at 10,000 rpm for 15 min at 4°C. The pellet was resuspended in 0.5% hexadecyltrimethyl ammonium bromide buffer (pH 5.4), and the samples were frozen and thawed 3 times in liquid nitrogen. Upon thawing, the samples were recentrifuged (10,000 rpm, 15 minutes, 4°C), and 25 μL of the supernatant were used for the MPO assay. The enzymatic reaction was assessed with 1.6 mM tetramethylbenzidine, 80 mM NaPO4, and 0.3 mM hydrogen peroxide. The absorbance was measured at 650 nm, and the results are expressed as optical density per milligram of tissue.
Determination of Interleukin-1β Levels in the Mouse Paw
Tissue levels of the proinflammatory cytokine interleukin (IL)-1β were measured according to the protocol described by da Cunha et al.14 The animals were treated with MAB (8.37, 27.93 or 83.79 μmol/kg, PO), and after 1 hour, they received a 50-μL i.d. injection of carrageenan (300 μg/paw) into the right paw. The mice were killed 6 hour after the carrageenan injection. Another group of mice received saline into the right hindpaws and were used as a control. Tissues were placed in phosphate-buffered saline (pH = 7.4; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4), containing 0.4 M NaCl, 0.1 M PMSF, 10 mM EDTA, 0.05% Tween 20, 0.5% bovine serum albumin, and 2 mg/mL of aprotinin, homogenized, centrifuged at 3000 × g for 10 min and stored at −20°C until further analysis. Cytokine levels were evaluated using an ELISA kit, according to the manufacturer's recommendations (R&D Systems).
Measurement of Nonspecific Effects
To exclude possible nonspecific effects of MAB on locomotor activity, mice were treated with MAB (83.79 μmol/kg, PO), and after 1 hour, locomotor activity was assessed in the open-field test, as described previously.15 The number of squares crossed with all paws (“crossings”) was counted in a 6-minute session.
The interference of MAB on motor coordination was also evaluated.16 This test was performed using a horizontal rota-rod device (Letica Scientific Instrument, Barcelona, Spain) set to rotate at 22 rpm. Mice that were able to remain on the apparatus for >60 seconds were selected and treated with MAB (83.79 μmol/kg, PO) 1 hour before the test. The control group received vehicle (10 mL/kg). The time (in seconds) until mice fell off the rota-rod (in a total period of 60 seconds) was recorded.
Rectal temperature was measured using a digital thermometer (B & D, Frankin,NJ). The probe (2 mm diameter) was dipped into semisolid paraffin before insertion and held in the rectum for 20 seconds until steady readings were obtained. The mice were treated with MAB (83.79 μmol/kg, PO) 1 hour before the temperature measurement.
We also investigated the interference of MAB (8.37, 27.93, or 83.79 μmol/kg, PO) in the mechanical and thermal sensitivity in the absence of pathology. The mechanical threshold evaluation consists of evoking a hindpaw flexion reflex with a hand-held force transducer (electronic anaesthesiometer; IITC Life Science, Woodland Hills, CA), adapted with a 0.5-mm2 polypropylene tip.17 After paw withdrawal, the intensity of the pressure was automatically recorded, and the final response value was obtained by averaging 3 measurements. The animals were tested before and after treatments. For evaluating thermal sensitivity in healthy animals, the mice were previously tested on the hotplate, maintained at 50 ± 1°C, 24 hours before the treatment.18 Subsequently, MAB was administered, and after 1 hour, mice were tested again on the hotplate test. Morphine was used as a positive-control.
Drugs and Reagents
The following reagents and drugs were used: gabapentin (Neurontin®; Park-Davis, Brazil); carrageenan, CFA (Sigma Aldrich, St Louis, MO), chloral hydrate (Vetec; Brazil), and Tween 80 (Merck, Germany). Morphine (Dimorf®) was kindly provided by Cristália, Brazil. MAB was diluted in saline (0.9%) plus Tween 80 (the final concentration did not exceed 0.5%, which when given alone, had no effect on MAB responses) and control animals were treated with this vehicle. The doses of indomethacin and gabapentin were selected on the basis of literature data or pilot experiments.10
The results are presented as the mean ± SEM of 5 to 7 animals, except for the ID50 values (i.e., the dose of MAB that reduced the nociceptive responses by 50% relative to the control values), which are presented as the means accompanied by their respective 95% confidence limits. The ID50 values were determined by the least squares method. For hypersensitivity methods, ID50 values were based on the area under curve of each group. The percentages of inhibition are reported as the mean ± SEM of inhibitions obtained for each individual experiment. Statistical comparisons between the data obtained from MAB-treated group and vehicle-treated group were performed by 1-way ANOVA, followed by Newman-Keuls' test, or by a 2-way ANOVA, followed by Bonferroni's test. P values <0.05 (P < 0.05 or less) were considered significant.
Figure 2 (A and B) demonstrates that the i.d. injection of carrageenan (300 μg/paw) significantly reduced the mechanical sensitivity threshold (P < 0.001) when compared to basal thresholds. Oral treatment with MAB (8.37, 27.93, or 83.79 μmol/kg) reduced the mechanical hypersensitivity induced by i.d. injection of carrageenan, with inhibition of 22% ± 6%, 45% ± 5%, and 69% ± 3%, respectively, and an ID50 value of 46.6 (35.6 to 61.0) μmol/kg. Indomethacin, a nonsteroidal anti-inflammatory drug used as positive control, was also capable of inhibiting the mechanical sensitization induced by carrageenan, with an inhibition of 58% ± 9%.
However, when hypersensitivity was induced by an i.d. injection of CFA, the pretreatment with MAB at a dose of 27.93 μmol/kg slightly reduced mechanical hypersensitivity with inhibition of 21% ± 4% (Fig. 2, C and D), whereas gabapentin was able to increase the mechanical withdraw threshold, inhibiting 92% ± 3%, for up to 48 hours.
To evaluate its therapeutic effect, MAB was administered 24 hours after the induction of hypersensitivity by CFA injection, twice daily for 5 days. As demonstrated in Figure 3 (A and B), the mice treated orally with MAB (83.79 μmol/kg) had a slight reduction in mechanical hypersensitivity when compared to the control group, with an inhibition of 35% ± 5%. This effect was observed for up to 2 hours after treatment (Fig. 3A). No effects were observed with the highest and the lowest doses of MAB. Gabapentin, a drug used clinically to treat chronic pain states, abolished the sensitization induced by CFA (75% ± 6%). Injection of CFA into 1 hindpaw of a mouse also produces contralateral hypersensitivity, a mechanism that involves modulation of the pain pathways in the spinal cord. In contrast to what was observed in the ipsilateral hindpaw, MAB (83.79 μmol/kg, PO) or gabapentin (0.4 mmol/kg, PO) was capable of preventing the onset of mechanical hypersensitivity in the contralateral hindpaw, with inhibitions of 73% ± 8% and 95% ± 4%, respectively (Fig. 3, C and D).
The antihypersensitivity effect of MAB on neuropathic pain-like behavior was also evaluated. In this experiment mice were treated twice daily for 5 days beginning 4 days after PLSN surgery. MAB (83.79 μmol/kg, PO) was markedly effective in reducing the mechanical hypersensitivity induced by PLSN (Fig. 4, A and B). The effect obtained with oral MAB treatment was maintained throughout the period of evaluation, with inhibition of 62% ± 10%. Gabapentin-treated mice (0.4 mmol/kg, PO) also demonstrated a marked decrease in mechanical hypersensitivity (71% ± 3%). To evaluate the thermal sensitivity of PLSN-treated mice, the plantar test was used. Figure 5A demonstrates that operated mice had decreased thermal withdrawal threshold when compared to the sham-operated group (P < 0.001). Similar to what was observed in the evaluation of mechanical hypersensitivity, mice treated orally with MAB (8.37 μmol/kg) showed inhibition of thermal sensitization for up to 11 days after surgery. MAB (83.79 μmol/kg, PO) or gabapentin (0.4 mmol/kg, PO) was also effective in reversing the cold hypersensitivity induced by PLSN for up to 24 hour after the treatment, with inhibitions of 98% ± 1% and 99% ± 1%, respectively (Fig. 5, B and C).
To verify the possible sites of action, MAB was administered by IV, i.d., i.t., or i.c.v. routes. Figure 6B demonstrates that IV administration of MAB (0.83, 2.79, or 8.37 μmol/kg) reduced mechanical hypersensitivity induced by carrageenan, with inhibition of 40% ± 2%, 22% ± 4%, and 30% ± 5%, respectively. Furthermore, when MAB was injected i.t., it inhibited the mechanical hypersensitivity by 47% ± 4%, 79% ± 6%, and 21% ± 9% for doses 8.3, 27.93, and 83.79 pmol/kg, respectively (Fig. 6, E and F). The administration of MAB by i.d. or i.c.v. routes was ineffective in interfering with the hypersensitivity induced by carrageenan (Fig. 6, C, D, G, and H).
The results in Figure 7A indicate that the carrageenan-induced MPO activity increase was significantly inhibited by treatment with MAB (8.37, 27.93, or 83.79 μmol/kg, PO, 1 hour prior), when compared to the control group (79% ± 4%, 81% ± 2%, and 79% ± 3%, respectively). The i.d. injection of carrageenan (300 μg/paw) significantly increased the IL-1β levels (Fig. 7 B) in the mouse hindpaw tissue. Systemic treatment with MAB (83.79 μmol/kg, PO) markedly reduced the enhancement of IL-1β (Fig. 7 B), which was similar to the results obtained with the positive control dexamethasone. The observed inhibitions were 64% ± 14% and 52% ± 7% for MAB and dexamethasone, respectively.
MAB (83.79 μmol/kg, PO) or gabapentin (0.4 mmol/kg, PO), administered 1 hour before the tests, did not affect the locomotor activity, motor coordination, or body temperature (Table 1). Importantly, no significant differences were observed in the mechanical or thermal withdrawal thresholds in mice treated with MAB, suggesting that MAB does not interfere with the sensitivity or the nociceptive threshold of healthy animals (Fig. 8, A and B).
In the present study, it was demonstrated that oral administration of MAB had significant antihypersensitivity effects in different experimental models of inflammatory and neuropathic pain in mice. These effects were similar to those of drugs used clinically, such as indomethacin and gabapentin.
Our study demonstrated the effect of MAB in mechanical hypersensitivity induced by i.d. injection of carrageenan in mice. Carrageenan is an inflammatory agent largely used as a pharmacological tool for investigating inflammatory hyperalgesia in rats and mice.19 When injected into the plantar surface of the animal's hindpaw, carrageenan causes inflammation, which results in hyperalgesia.20 Tissue injury, resulting from the injection of carrageenan, causes the release of different chemical mediators, including prostaglandinE2,21 mast cell products (histamine and serotonin22), neuropeptides,23 and cytokines (tumor necrosis factor α and IL-1β20).
This study demonstrated that oral treatment with MAB reduces the mechanical hypersensitivity induced by carrageenan in mice. This effect was greatest in the first hour after carrageenan injection, and it persisted for up to 72 hours after the injection. It is important to note that, when compared with indomethacin, MAB had a more persistent antihypersensitive effect, although the area under curve of both substances is similar. These results suggest that MAB may be interfering with different pathways involved in inflammatory pain signaling.
The effect of MAB on inflammatory hypersensitivity induced by i.d. injection of CFA was also investigated. This model closely parallels human persistent inflammatory diseases, such as rheumatoid arthritis. The inflammatory response induced by CFA develops rapidly and may persist for a few weeks or months. The hypersensitivity is mediated by local sensitization of nociceptors, involves immune response, and changes in the central nervous system.24 The effect of MAB was evaluated using 2 treatment protocols, preventive and therapeutic. MAB (27.93 μmol/kg, PO), when administered before the induction of hypersensitivity, was capable of interfering with mechanical hypersensitivity only at 4 hours after CFA injection. In contrast, when MAB was dosed after the injection of CFA, twice a day, for up to 5 days, a reduction in the hypersensitivity on the fifth day after the MAB treatment, with the higher dose (83.79 μmol/kg), was observed. I.d. injection of CFA produces mechanical and thermal sensitization in the contralateral hindpaw, an event that involves central reorganization (reorganization of spinal cord neurons).10 MAB abolished the mechanical hypersensitivity induced by CFA in the contralateral hindpaw, suggesting that this compound may interfere with the central nociceptive pathways.
The PLSN model was used to evaluate the antihypersensitivity effect of MAB in neuropathic pain. After PLSN, both mechanical and thermal (cold and heat) sensitivity were evaluated. Oral treatment with MAB was able to inhibit the mechanical hypersensitivity induced by nerve injury; this inhibition was sustained for up to 8 days after the surgery. Previous data have shown that the hypersensitivity response induced by PLSN has rapid onset and persists for approximately 2 weeks after nerve injury.25 PLSN has also been shown to reduce the response latency for thermal stimuli (<4°C or >40°C), without interfering with the receptive fields for mechanical and thermal stimuli.26 MAB significantly inhibited thermal hypersensitivity (cold and heat) for up to 24 hours after the treatment. The antihypersensitivity effect of MAB was similar to that observed with gabapentin. Initially, gabapentin was introduced as a γ-aminobutyric acid A analog. However, Li et al.27 demonstrated that it also interacts with the α2δ subunit of the calcium channel. Gabapentin is currently the drug of choice for the clinical treatment of chronic pain.
To evaluate the site of action of MAB, it was administered by peripheral (IV or intraplantar) and central (i.t. or i.c.v.) routes. Our results suggest that the antihypersensitivity effects of MAB are due to its activity on the spinal pathways of pain control, once the i.t., but not i.c.v., administration was capable of interfering with hypersensitivity induced by carrageenan. Furthermore, MAB administered orally or IV may be acting in the central nervous system by crossing the blood-brain barrier (BBB), a possibility that requires further investigation. We did not determine whether the BBB is disrupted after the induction of hypersensitivity. Notwithstanding, if we consider that inflammation induced by carrageenan, CFA or PLSN is accompanied by cell migration and release of inflammatory mediators (such as bradykinin and substance P), it is feasible that the BBB permeability was altered, permitting the passage of MAB through the BBB. It is also important to mention that MAB probably did not require hepatic metabolism to act, as IV administration of MAB was capable of interfering with the hypersensitivity response induced by carrageenan.
Additionally, we demonstrated that systemic treatment with MAB significantly prevented neutrophil migration and reduced the production of IL-1β induced by carrageenan. It is tempting to suggest that the antinociceptive effects described for MAB in the carrageenan model are probably related to its ability to reduce cell migration and generate proinflammatory cytokines. Cunha et al.28 have also suggested that the release of primary mediators responsible for carrageenan-induced mechanical hypersensitivity is preceded by the production of a cascade of proinflammatory cytokines. Likewise, neutrophil migration appears to represent a pivotal step in the cascade of events leading to mechanical hypersensitivity evoked by carrageenan.29
The majority of drugs used clinically to treat chronic pain have action in the central nervous system, and their central action results in side effects, such as incoordination, sedation, and muscle weakness. Furthermore, the central action of clinically used drugs also represents the largest source of error in studies of drugs that interfere with the central nociceptive pathways.30 Of note, the antihypersensitivity caused by MAB or gabapentin, at these doses, was not associated with nonspecific effects, such as changes in locomotor activity or motor coordination. Furthermore, this compound does not seem to interfere with pain sensitivity (thermal or mechanical) or nociceptive thresholds in healthy mice, a frequent characteristic of clinically used analgesics, such as morphine.
In summary, we have demonstrated that MAB reduces the mechanical hypersensitivity induced by carrageenan, CFA, or PLSN in mice. This effect was observed when the compound was dosed preventively or therapeutically, systemically or by spinal routes: (1) suggesting the influence in the beginning of pain signaling and in the established pain process; and (2) suggesting a central activity of the compound. Moreover, our results implicate the involvement of cell migration and cytokine production in the antihypersensitivity effects obtained with MAB. These findings might have additional therapeutic implications for the development of a new drug to treat persistent pain without significant side effects.
Name: Carla de Souza Antonialli, MSc.
Contribution: This author helped conduct the study.
Attestation: Carla de Souza Antonialli has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Gislaine Francieli da Silva, MSc.
Contribution: This author helped conduct the study.
Attestation: Gislaine Francieli da Silva has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Lilian Wunsch Rocha, MSc Student.
Contribution: This author helped conduct the study.
Attestation: Lilian Wunsch Rocha has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Elis R. Monteiro.
Contribution: This author helped conduct the study.
Attestation: Elis R. Monteiro has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Márcia Maria de Souza, PhD.
Contribution: This author helped analyze the data.
Attestation: Márcia Maria de Souza has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Ângela Malheiros, PhD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Attestation: Ângela Malheiros has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Rosendo Augusto Yunes, PhD.
Contribution: This author helped design the study.
Attestation: Rosendo Augusto Yunes has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Nara Lins Meira Quintão, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Nara Lins Meira Quintão has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
This manuscript was handled by: Quinn H. Hogan, MD.
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