Activation of central a2-adrenoceptors mediates salivarygland vasoconstriction

Thiago S. Moreira a,*, Ana C. Takakura b, Jose V. Menani c, Eduardo Colombari c

aDepartment of Physiology and Biophysics, Institute of Biomedical Science, University of Sao Paulo, 05508-000, Sao Paulo, BrazilbDepartment of Pharmacology, Institute of Biomedical Science, University of Sao Paulo, 05508-900, Sao Paulo, BrazilcDepartment of Physiology and Pathology, School of Dentistry, Sao Paulo State University, 14801-903, Araraquara, Brazil

a r c h i v e s o f o r a l b i o l o g y 5 8 ( 2 0 1 3 ) 1 6 7 – 1 7 3

a r t i c l e i n f o

Article history:

Accepted 24 June 2012

Keywords:

a2-Adrenergic receptor

Pilocarpine

Parasympathetic

Blood flow and vascular resistance

Salivary gland

Moxonidine

a b s t r a c t

Objective: Peripheral treatment with the cholinergic agonist pilocarpine increases salivary

gland blood flow and induces intense salivation that is reduced by the central injection of

moxonidine (a2-adrenoceptors/imidazoline agonist). In the present study, we investigated

the effects of the intracerebroventricular (i.c.v.) injection of pilocarpine alone or combined

with moxonidine also injected i.c.v. On submandibular/sublingual gland (SSG) vascular

resistance. In addition, the effects of these treatments on arterial pressure, heart rate and on

mesenteric and hindlimb vascular resistance were also tested.

Design: Male Holtzman rats with stainless steel cannula implanted into lateral ventricle and

anaesthetized with urethane + a-chloralose were used.

Results: Pilocarpine (500 nmol/1 ml) injected i.c.v. Reduced SSG vascular resistance and

increased arterial pressure, heart rate and mesenteric vascular resistance. Contrary to

pilocarpine alone, the combination of moxonidine (20 nmol/1 ml) and pilocarpine injected

i.c.v. Increased SSG vascular resistance, an effect abolished by the pre-treatment with the

a2-adrenoceptor antagonist yohimbine (320 nmol/2 ml). The increase in arterial pressure,

heart rate and mesenteric resistance was not modified by the combination of moxonidine

and pilocarpine i.c.v.

Conclusion: These results suggest that the activation of central a2-adrenoceptors may

oppose to the effects of central cholinergic receptor activation in the SSG vascular resis-

tance.

# 2012 Elsevier Ltd. All rights reserved.

Available online at www.sciencedirect.com

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1. Introduction

Pilocarpine is a muscarinic cholinergic agonist used to reduce

dryness of the oral mucosa in patients affected by salivary

gland diseases.1,2 It is well accepted that pilocarpine stimu-

lates salivary secretion by acting on cholinergic receptors in

the salivary gland.1,2 This idea is supported by the sialogogue

effect of pilocarpine in isolated salivary glands.3

* Corresponding author at: Department of Physiology and Biophysics,Lineu Prestes, 1524, 05508-000, Sao Paulo, Brazil. Tel.: +55 11 3091 776

E-mail address: tmoreira@icb.usp.br (T.S. Moreira).

0003–9969/$ – see front matter # 2012 Elsevier Ltd. All rights reservehttp://dx.doi.org/10.1016/j.archoralbio.2012.06.017

However, recent evidence suggests that peripheral admin-

istered pilocarpine can also activate muscarinic receptors in

the brain to stimulate salivation.4,5,6 The suggestion that

pilocarpine may act centrally to stimulate salivary secretion is

also reinforced by studies that have shown that salivation

induced by pilocarpine injected peripherally is reduced by

focal lesions in the forebrain.7–9 Pilocarpine injected periph-

erally also induces submandibular/sublingual gland (SSG)

vasodilation,10 an effect due to the direct action of pilocarpine

Institute of Biomedical Science, University of Sao Paulo, Av. Prof4; fax: +55 11 3091 7285.

d.

a r c h i v e s o f o r a l b i o l o g y 5 8 ( 2 0 1 3 ) 1 6 7 – 1 7 3168

in the salivary glands and perhaps also to the activation of

central mechanisms.6

Moxonidine (a2-adrenoceptor/imidazoline receptor ago-

nist) is an anti-hypertensive drug that acts centrally to reduce

sympathetic nerve discharge.11–14 Moxonidine injected i.c.v.

reduces peripheral pilocarpine-induced salivation and vaso-

dilation in the SSG.4,10,15 The reduction of pilocarpine-induced

salivation produced by moxonidine injected i.c.v depends on

the activation of central a2-adrenoceptors,4,15 however, the

receptor subtypes involved in the moxonidine inhibition of

pilocarpine-induced SSG vasodilation have not been charac-

terized.

Therefore, in the present study we investigated the effects

of i.c.v. injection of pilocarpine alone or combined with i.c.v.

moxonidine on SSG, mesenteric and hindlimb blood flow and

vascular resistance, mean arterial pressure (MAP) and heart

rate (HR). Additionally, we also investigated the effects of

yohimbine (a2-adrenoceptor antagonist) injected i.c.v. com-

bined with moxonidine and pilocarpine i.c.v. on MAP, HR and

SSG, mesenteric and hindlimb blood flow and vascular

resistance.

2. Methods

2.1. Animals

Male Holtzman rats weighing 300–350 g were used. The

animals were housed individually in stainless steel cages in

a room with controlled temperature (23 � 2 8C) and humidity

(55 � 10%). Lights were on from 7:00 am to 7:00 pm. Guabi rat

chow (Paulınia, SP, Brazil) and tap water were available

ad libitum. The experimental protocols were approved by the

Animal Experimentation Ethics Committee of the Federal

University of Sao Paulo.

2.2. Brain surgery

Rats were anaesthetized with intraperitoneal (i.p.) injection of

ketamine (80 mg/kg of body wt) combined with xylazine (7 mg/

kg of body wt) and placed in a stereotaxic frame (model 900,

David Kopf Instruments). The skull was levelled between

bregma and lambda. A stainless steel cannula

(10 mm � 0.6 mm o.d.) was implanted into the lateral cerebral

ventricle (LV) using the following stereotaxic coordinates:

0.3 mm caudal to bregma, 1.5 mm lateral to midline and

3.6 mm below the dura mater. The cannula was fixed to the

cranium with dental acrylic resin and jeweller screws. Rats

received a prophylactic dose of penicillin (30,000 IU) given

intramuscularly and a subcutaneous injection of the analgesic

Ketoflex (ketoprofen 1%, 0.03 ml/rat) post-surgically. After the

surgery, the rats were maintained in individual box with

free access of tap water and food pellets for at least 7 days

before the tests.

2.3. Drugs

Moxonidine hydrochloride (20 nmol/1 ml), a gift from Solvay

Pharma (Germany), pilocarpine hydrochloride (500 nmol/

1 ml) and yohimbine hydrochloride (320 nmol/2 ml) from

Sigma Chemical Co., USA were injected i.c.v. A mix of

propylene glycol/water 2:1 was used as vehicle for yohimbine

and moxonidine because these drugs at the doses used are

not soluble in saline. Pilocarpine was dissolved in isotonic

saline.

The dose of pilocarpine used in the present study was

based on a previous study employing pilocarpine i.c.v. to

induce salivation in rats.7 The doses of yohimbine and

moxonidine were based on previous studies that have shown

the effects of different doses of yohimbine and moxonidine on

pilocarpine-induced salivation, water and sodium intake and

cardiovascular responses.4,16,17

2.4. Cerebral injections

Injections into the LV were made using 10 ml Hamilton

syringes connected by polyethylene tubing (PE-10) to injec-

tion cannulas 2 mm longer than the guide cannulas

implanted into the brain. The volume injected into the LV

was 1 or 2 ml.

2.5. Arterial pressure and heart rate recordings

On the day of the experiment rats were anaesthetized with

urethane (1.2 g/kg of body weight i.v.) and a-chloralose

(60 mg/kg of body weight i.v.) (after the induction with 1%

halothane in 100% O2). A femoral artery catheter (PE-10

connected to PE-50) was implanted for the record of pulsatile

arterial pressure, mean arterial pressure (MAP) and heart rate

(HR). A femoral vein catheter was implanted for administra-

tion of anaesthetic. To record pulsatile arterial pressure, MAP

and HR, the arterial catheter was connected to a Statham

Gould (P23 Db) pressure transducer coupled to a pre-amplifier

(model ETH-200 Bridge Bio Amplifier, CB Sciences) and to a

Powerlab computer recording system (model Powerlab 16SP,

ADInstruments). Recordings began 10 min after the connec-

tion of the arterial line to the pressure transducer. MAP and

HR were continuously recorded during 1 h and were analysed

at every 5 min. Baseline values were recorded for 10 min and

were analysed immediately before yohimbine or vehicle

injection (first treatment). These values were used as

reference to calculate the changes produced by the treat-

ments.

2.6. Submandibular/sublingual gland, superiormesenteric and low abdominal aorta artery blood flowrecordings

Immediately after vein and artery catheterization, an incision

was made in ventral midline of the neck to localize the right

submandibular/sublingual gland (SSG) complex and the artery

that irrigates the SSG complex. The SSG artery, a cervical

branch of external maxillary artery is usually larger just above

the anterior margin of posterior belly of digastricus.6,10 The

artery that irrigates the SSG complex was isolated and a

miniature pulsed Doppler flow probe (Iowa Doppler Products;

Iowa City, IA) was perfectly adjusted around the artery to

record blood flow.

A midline laparotomy was also performed and miniature

pulsed Doppler flow probes were placed around the superior

a r c h i v e s o f o r a l b i o l o g y 5 8 ( 2 0 1 3 ) 1 6 7 – 1 7 3 169

mesenteric (SM) artery and the low abdominal aorta for

measurement of mesenteric and hindlimb blood flow,

respectively. The probes were fixed to the surrounding tissues

with suture thread. Data from animals in which the probes

moved during the experiment were not considered for

analysis.

The flow probes were connected to a Doppler flowmeter

(Dept of Bioengineering, University of Iowa, Iowa City, IA)

coupled to a Powerlab computer record system (model

Powerlab 16SP, ADInstruments) for blood flow recording.

Details of the Doppler flow recording technique, including the

reliability of the method for estimation of flow velocity, have

been described previously.18 Relative SSG, mesenteric and

hindlimb vascular resistance changes were calculated as the

ratio of MAP and Doppler shift.

Blood flow was continuously recorded for 1 h. Blood flow

and vascular resistance were analysed for every 5 min.

Baseline values were recorded for 10 min and were analysed

immediately before yohimbine or vehicle injection (first

injection). The values were used as reference to calculate

the changes produced by the treatments.

2.7. Histology

After the experiments, Evans blue (100 nl of a 2% solution) was

microinjected into the LV for histological analysis. The animals

were deeply anaesthetized with urethane (1.2 g/kg of body

weight i.v.) and a-chloralose (60 mg/kg of body weight i.v.).

Saline followed by 10% buffered formalin was perfused through

the heart. The brains were frozen, cut coronally into 50 mm

sections and stained with Giemsa stain. Only animals with

injections into the LV were considered for statistical analysis.

Fig. 1 – Changes in (A) blood flow and (B) vascular resistance in

pilocarpine (500 nmol/1 ml) injected i.c.v. in rats pre-treated wit

moxonidine (20 nmol/1 ml) or vehicle injected i.c.v. The results

*Different from vehicle + vehicle + saline (Student–Newman–Keu

2.8. Statistical analysis

All values were expressed as means � SEM. Statistical

analysis was performed using two-way analysis of variance

(ANOVA) with repeated measures followed by Student–

Newman–Keuls post hoc tests to determine significant

differences between groups. Significance level was set at

p < 0.05.

2.9. Experimental protocols

All studies were performed in rats anaesthetized with

urethane (1.2 g/kg of body weight i.v.) and a-chloralose

(60 mg/kg of body weight i.v.). After 10 min of control

(baseline) recording of MAP, HR and blood flow velocity in

SSG, SM and abdominal aorta arteries, yohimbine (320 nmol/

2 ml) or vehicle was injected i.c.v. Moxonidine (20 nmol/1 ml)

or vehicle was injected i.c.v. 15 min after central injection of

yohimbine or vehicle. Pilocarpine (500 nmol/1 ml) or saline

was injected i.c.v. 15 min after the i.c.v. injection of

moxonidine or vehicle. The recordings stopped 30 min after

the last injection.

To study the involvement of central a2-adrenoceptor

on the association of cardiovascular effects of central

moxonidine and pilocarpine, 4 groups of rats were used:

(1) a control group that received vehicle i.c.v. followed by

vehicle and saline i.c.v.; (2) a group injected with

yohimbine i.c.v. followed by moxonidine and pilocarpine

i.c.v.; (3) a group treated with vehicle i.c.v. Followed by

moxonidine and pilocarpine i.c.v.; (4) a group that

received vehicle i.c.v. Followed by vehicle and pilocarpine

i.c.v.

the submandibular/sublingual gland (SSG) produced by

h yohimbine (320 nmol/2 ml) or vehicle combined with

are represented as means W SEM. n = number of rats.

ls test, p < 0.05).

a r c h i v e s o f o r a l b i o l o g y 5 8 ( 2 0 1 3 ) 1 6 7 – 1 7 3170

3. Results

3.1. Changes in the SSG vascular resistance and bloodflow produced by pilocarpine i.c.v. in rats pre-treated withmoxonidine i.c.v. alone or combined with yohimbine i.c.v.

Pilocarpine (500 nmol/1 ml) injected i.c.v. reduced SSG vascular

resistance (�34 � 11%, vs. saline: 5 � 5%) [F (3, 17) = 118,13;

p < 0.01] and increased SSG blood flow (43 � 18%, vs. saline:

6 � 3%) [F (3, 17) = 105,66; p < 0.01] (Fig. 1).

Contrary to the effects of pilocarpine injected i.c.v. alone,

the SSG vascular resistance increased (80 � 36%) and the SSG

blood flow was reduced (�45 � 15%) by the treatment with

pilocarpine i.c.v. combined with moxonidine (20 nmol/1 ml)

i.c.v. (Fig. 1).

The pre-treatment with yohimbine (320 nmol/2 ml) injected

i.c.v. abolished the increase in SSG vascular resistance (3 � 6%,

vs: moxo + pilo: 80 � 36%) and the vasodilatation (7 � 13%, vs:

moxo + pilo: �45 � 15%) produced by combining moxonidine

and pilocarpine i.c.v. (Fig. 1).

3.2. Changes in MAP and HR and in the vascularresistance and blood flow in the SM artery and hindlimbproduced by pilocarpine injected i.c.v. in rats pre-treated withmoxonidine i.c.v. alone combined with yohimbine i.c.v.

Pilocarpine (500 nmol/1 ml) injected i.c.v. induced pressor

responses (21 � 4 mmHg, vs. saline: 2 � 2 mmHg) [F (3,

17) = 63,47; p < 0.05] and tachycardia (15 � 4 bpm, vs. vehicle

3 � 4 bpm) [F (3, 17) = 44,12; p < 0.05] and increased vascular

resistance (28 � 4% vs. saline: 6 � 3%) [F (3, 17) = 46,19;

Fig. 2 – Changes in (A) mean arterial pressure (MAP) and (B) hea

1 ml) injected i.c.v. in rats pre-treated with yohimbine (320 nmo

or vehicle injected i.c.v. The results are represented as means W

vehicle + vehicle + saline (Student–Newman–Keuls test, p < 0.05

p < 0.05] and reduced blood flow in the SM artery

(�13 � 6% vs. saline: 2 � 1%) [F (3, 17) = 53,07; p < 0.05],

without changing hindlimb vascular resistance or blood

flow (Figs. 2–4).

Prior injection of moxonidine (20 nmol/1 ml) i.c.v. alone or

combined with yohimbine (320 nmol/2 ml) did not modify

the pressor response (18 � 4 and 16 � 3 mmHg, respectively),

thetachycardia (12 � 4and13 � 3 bpm,respectively), theincrease

in SM vascular resistance (20 � 4% and 19 � 4%, respectively) and

the reduction of blood flow (�10 � 4% and �12 � 3%, respectively)

produced by i.c.v. pilocarpine (Figs. 2 and 3).

The baseline MAP and HR immediately before yohimbine or

vehicle injections in each group of rats are presented in

Table 1.

4. Discussion

The present results show that central injections of pilocar-

pine reduce SSG vascular resistance and the increase MAP,

HR and mesenteric vascular resistance. Contrary to the

reduction in the salivary gland vascular resistance, the

combination of moxonidine and pilocarpine injected i.c.v.

increased SSG vascular resistance, an effect abolished by the

previous injection of yohimbine i.c.v. The changes in

mesenteric vascular resistance, MAP and HR produced by

pilocarpine i.c.v. were not altered by the central injection of

moxonidine. Hindlimb vascular resistance was not affected

by either treatment. These results suggest that the activation

of central a2-adrenoceptors may oppose to the effects of

central cholinergic receptor activation in the SSG vascular

resistance.

rt rate (HR) produced by produced by pilocarpine (500 nmol/

l/2 ml) or vehicle combined with moxonidine (20 nmol/1 ml)

SEM. n = number of rats. *Different from

).

Fig. 3 – Changes in (A) blood flow and (B) vascular resistance in superior mesenteric (SM) artery produced by pilocarpine

(500 nmol/1 ml) injected i.c.v. in rats pre-treated with yohimbine (320 nmol/2 ml) or vehicle combined with moxonidine

(20 nmol/1 ml) or vehicle injected i.c.v. The results are represented as means W SEM. n = number of rats. *Different from

vehicle + saline (Student–Newman–Keuls test, p < 0.05).

a r c h i v e s o f o r a l b i o l o g y 5 8 ( 2 0 1 3 ) 1 6 7 – 1 7 3 171

The effects produced by i.c.v. injection of pilocarpine on

MAP, HR and on SSG and mesenteric resistances were

similar to those produced by peripheral injections of

pilocarpine, which reinforces the suggestion that pilocar-

pine injected peripherally may act centrally to reduce SSG

Fig. 4 – Changes in (A) blood flow and (B) vascular resistance in

i.c.v. in rats pre-treated with yohimbine (320 nmol/2 ml) or vehi

injected i.c.v. The results are represented as means W SEM. n =

Newman–Keuls test, p < 0.05).

vascular resistance and to increase MAP, HR and mesen-

teric vascular resistance.6,10 In addition to the central

effects, pilocarpine injected peripherally may also produce

SSG vasodilation by acting directly in the salivary glands.

In spite of this direct effect on salivary glands, moxonidine

hindlimb produced by pilocarpine (500 nmol/1 ml) injected

cle combined with moxonidine (20 nmol/1 ml) or vehicle

number of rats. *Different from vehicle + saline (Student–

Table 1 – Baseline MAP and HR in the groups of rats that received pilocarpine or saline injected i.c.v. after the pre-treatment with yohimbine or vehicle combined with moxonidine or vehicle injected i.c.v.

Treatments Baseline MAP (mmHg) Baseline HR (bpm)

Vehicle + vehicle + saline (i.c.v.) (n = 6) 102 � 4 382 � 9

Vehicle + vehicle + pilocarpine (i.c.v.) (n = 7) 100 � 3 373 � 10

Vehicle + moxonidine + pilocarpine (i.c.v.) (n = 7) 101 � 4 388 � 12

Yohimbine + moxonidine + pilocarpine (i.c.v.) (n = 8) 103 � 4 380 � 11

The results are represented as means � SEM. n = number of rats. Pilocarpine (500 nmol/1 ml); moxonidine (20 nmol/1 ml); yohimbine (320 nmol/

2 ml).

a r c h i v e s o f o r a l b i o l o g y 5 8 ( 2 0 1 3 ) 1 6 7 – 1 7 3172

injected i.c.v. combined with pilocarpine injected intrave-

nously also increased SSG vascular resistance,10 similar to

the effects of moxonidine combined with pilocarpine i.c.v.

(present results). Moxonidine injected i.c.v. alone also

increases SSG vascular resistance,10 which suggests that

the activation of central a2-adrenoceptors overcomes the

effects central cholinergic activation resulting in increased

SSG vascular resistance when pilocarpine is combined with

moxonidine both injected i.c.v.

The importance and the involvement of the central a2-

adrenoceptors in the inhibition of salivation were shown

previously by injecting clonidine intracisternally in cats that

received electrical stimulation of brainstem parasympathetic

nuclei.19 The effect of clonidine was inhibited by prior

intracisternal injection of yohimbine. Previous studies also

showed that the activation of central a2-adrenoceptors with

moxonidine reduces salivation and abolishes the increase in

the SSG blood flow produced by peripheral injection of

pilocarpine.4,10 The present results extend the conclusion of

the previous studies showing that: (1) central injection of

pilocarpine reduces SSG vascular resistance, similarly to the

activation of the parasympathetic output or to peripheral

injection of pilocarpine; (2) the increase in the SSG vascular

resistance after combining central injection of pilocarpine and

moxonidine results from the activation of central a2-adreno-

ceptor by moxonidine.

Pilocarpine at the dose used in the present study reduces

salivary gland vascular resistance and increases mesenteric

vascular resistance without changing hindlimb vascular

resistance. The opposite effects on vascular resistance suggest

that pilocarpine activates different mechanisms producing

specific adjustments in vascular resistance and blood flow in

different regions of the body. Pilocarpine injected centrally

produces:(1) decrease in the SSG vascular resistance and no

change in the hindlimb; (2) increase in MAP that seems to be

dependent on the increase in mesenteric vascular resistance;

(3) increase in HR that may be involved in pressor pathway. It

is well known that the central activation of cholinergic

receptors stimulates sympathetic activity and vasopressin

release, causing an increase in MAP.20,21 Combining the

increase in MAP and the reduced salivary gland vascular

resistance, central injection of pilocarpine produces a strong

increase in blood flow to the salivary glands and these effects

may explain why pilocarpine is so effective at inducing

salivary secretion.

Despite of the anti-hypertensive properties of moxonidine

(a2-adrenoceptor and imidazoline agonist12–14,24), no change in

pilocarpine-induced vasoconstriction and pressor responses

was observed following the treatment with moxonidine into

the lateral cerebral ventricle as previously demonstrated for

pilocarpine injected peripherally.10 Studies have demonstrat-

ed that moxonidine acts in the brainstem, particularly in

rostral ventrolateral medulla (RVLM), to reduce sympathetic

outflow and blood pressure.11–14 In the present study,

moxonidine was injected into the lateral cerebral ventricle

and in this case the main areas reached by moxonidine were

forebrain areas. The results suggest that the activity of the

cholinergic pressor mechanisms in the forebrain is not

modified by the activation of a2-adrenoceptors or imidazoline

receptors with i.c.v. injection of moxonidine.

The vasoconstriction in the salivary gland induced by

moxonidine may be the result of the activation of a

vasoconstrictor or removal of the vasodilator mechanism to

the salivary gland. The latter seems less likely because

vasodilator tone to the salivary glands is rather small.22

Whatever the mechanism activated by central moxonidine,

the increase in salivary gland vascular resistance produced by

i.c.v. moxonidine was not modified by peripheral10 and central

injection (present data) of pilocarpine.

It seems that pilocarpine acting centrally activates both

salivary gland secretion and vasodilation.7,8,10 Because saliva-

tion depends on secretory mechanisms and on the increase in

blood flow to the glands,23 reduction in salivation may occur if

one or both mechanisms are affected. The activation of a2-

adrenoceptor with moxonidine reduces the salivary secretion

and the vasodilation induced by pilocarpine.15,10 Therefore, it

is possible that moxonidine inhibits pilocarpine-induced

salivation at least partially by reducing salivary gland blood

flow. Besides this, the vasoconstriction and the reduction of

the blood flow to the salivary glands produced by the

activation of the central a2-adrenoceptors is probably impor-

tant for the sensation of dryness in the mouth by patients

treated with moxonidine or the same type of drugs.

In summary, the present results suggest that central

cholinergic and a2-adrenergic mechanisms have opposite

roles in the control of the salivary gland vascular resistance

and blood flow. However, the increase in MAP, HR and

mesenteric vascular resistance produced by the cholinergic

activation in the forebrain is not affected by central a2-

adrenoceptor activation, suggesting that different central

mechanisms are activated by pilocarpine to produce the

changes in the vascular resistance in different vascular

beds.

Funding

Sao Paulo State Foundation (FAPESP).

a r c h i v e s o f o r a l b i o l o g y 5 8 ( 2 0 1 3 ) 1 6 7 – 1 7 3 173

Competing interests

None declared.

Ethical approval

Experimental protocols were approved by the Animal Experi-

mentation Ethics Committee of the Federal University of Sao

Paulo (UNIFESP).

Acknowledgements

We would like to thank also Solvay Pharma and Dr. P.

Ernsberger for the donation of moxonidine. This research was

supported by public funding from Fundacao de Amparo a

Pesquisa do Estado de Sao Paulo (FAPESP) and Conselho

Nacional de Pesquisa (CNPq/PRONEX).

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