Spontaneously hypertensive rat as experimental model ofsalivary hypofunction

Daniele C.R. Picco a,*, Lilian F. Costa a, Alberto C.B. Delbem a, Kikue T. Sassaki b,Doris H. Sumida b, Cristina Antoniali b

aDepartment of Pediatrics and Social Dentistry, School of Dentistry of Aracatuba, Sao Paulo State University, UNESP, SP, BrazilbDepartment of Basic Sciences, School of Dentistry of Aracatuba, Sao Paulo State University, UNESP, SP, Brazil

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

a r t i c l e i n f o

Article history:

Accepted 22 July 2012

Keywords:

Hypertension

Salivary glands

Saliva

SHR

a b s t r a c t

Objective: To analyse the salivary activity in spontaneously hypertensive rats (SHRs) evalu-

ating biochemical parameters of saliva in 4-week-old and 12-week-old animals.

Design: Systolic blood pressure (SBP) was recorded by tail plethysmography. The salivary

flow rate was stimulated by pilocarpine (SFR). The pH and salivary buffering capacity (SBC)

were evaluated with a specific electrode. The concentrations of fluoride ([F�]) and calcium

([Ca++]) ions were determined using an electrode connected to a calibrated ion analyser. The

total protein concentration was determined by Lowry method, and amylase activity by

kinetic method. The salivary IgA was determined by enzyme-linked immunosorbent assay

(ELISA).

Results: The SFR, [F�] and [Ca++] increased with age in normotensive rats, however no

alteration in pH, total protein and IgA was observed between 4 and 12 weeks old Wistar

rats. SBC decreased with age in Wistar rats. The SFR was not altered between SHRs in

different ages and it was lower in 12 weeks old SHR when compared to Wistar rats. An

increase in the protein concentration, and in the amylase activity and [F�] was observed

with the development of SHR. Unaltered SBC, salivary IgA and [Ca++] were observed in 12

weeks old when compared to 4 weeks old SHR. The [Ca++] ions were reduced in saliva of SHR

than that of Wistar rats at 12 weeks. A lower pH was observed in saliva of Wistar than that of

SHR at 12 weeks.

Conclusions: SHR is an experimental model of salivary hypofunction, the decreased SFR

observed in SHR at different ages was associated to salivary biochemical parameter

alterations.

# 2012 Elsevier Ltd.

Available online at www.sciencedirect.com

journal homepage: http://www.elsevier.com/locate/aob

Open access under the Elsevier OA license.

1. Introduction

Saliva is an essential fluid for the maintenance of a healthy

oral mucosa. Patients with hyposalivation show a higher risk

of infections and carious lesions, impairing life quality. Many

studies have been performed to investigate the relationship

* Corresponding author at: Rua Edvard de Vita Godoy, 239, CEP: 05128E-mail address: dcrpicco@gmail.com (Daniele C.R. Picco).

0003–9969 # 2012 Elsevier Ltd.

http://dx.doi.org/10.1016/j.archoralbio.2012.07.008

Open access under the Elsevier OA license.

between hyposalivation and certain disease, such as hyper-

tension. Experimental studies1,2 have demonstrated signifi-

cant reduction in the salivary gland activity in the

spontaneously hypertensive rat (SHR), the most commonly

studied model of essential hypertension. We have reported3,4

a reduced salivary flow rate (SFR) and total salivary protein

concentration in young 4-week-old SHR. These results

-190, Sao Paulo, SP, Brazil. Tel.: +55 11 2892 1929.

a r c h i v e s o f o r a l b i o l o g y 5 7 ( 2 0 1 2 ) 1 3 2 0 – 1 3 2 6 1321

suggested that the alterations in the salivary activity observed

in SHR could not be associated only with the high levels of

arterial pressure. The aim of this study was to evaluate the

effects of growth/development (4 and 12-week-old) and age-

related hypertension (pre-hypertensive and hypertensive rats)

on saliva output and composition.

2. Materials and methods

2.1. Animals

All experiments conducted in this study were approved by the

Institutional Animal Research Ethics Committee (CEEA)

(process number 003176). Forty male rats divided into four

groups of 10 animals were used: (1) 4-week-old SHR, (2) 12-

week-old SHR, (3) 4-week-old Wistar, and (4) 12-week-old

Wistar. The animals were kept in an environment with

controlled temperature (22–24 8C) and light cycle (12 h/light

and 12 h/darkness), receiving standard food and water

‘‘ad libitum’’. Systolic blood pressure (SBP) of SHR and Wistar

rats was recorded by tail plethysmography (Plethysmograph

Physiograph1 MK-III-S/NBS, Narco Bio-Systems, TX, USA).

Only 12-week-old Wistar rats with SBP of approximately

112 mmHg and 12-week-old SHRs with SBP equal to or higher

than 150 mmHg were used in the experiments.

2.2. Saliva collections and salivary flow

After 12-h fasting, rats were anaesthetized with ketamine

(45 mg/kg, im) and xylazine (5 mg/kg, im) and the salivary flow

was stimulated by pilocarpine nitrate (5 mg/kg BW, ip, Sigma,

MO, USA). Saliva collection was performed according to

Bernarde’s method.5 After the pilocarpine injection, the

animals were placed in an inclined bed. The stimulated saliva

was collected in flasks kept on ice for 15 min after the first

drop, in temperature-controlled room (20 8C). The saliva

volume was calculated from the difference in weight of full

and empty flasks, considering the saliva density as 1 mg/mL.

As we observed that rat body weight was altered at different

ages, the SFR was normalized and expressed as mL/min/100 g

body weight. Saliva samples were stored in tubes at �70 8C

until biochemical experiments were conducted.

2.3. pH and salivary buffering capacity

The pH and salivary buffering capacity (SBC) were evaluated in

fresh saliva. Immediately after collection, the salivary pH was

measured in saliva samples (200 mL) with a specific electrode

(Analyzer) connected to a pH meter (Thermo Fischer, Orion

720A, MA, USA), previously calibrated. The SBC was calculated

by titulometric method, according to the volume of lactic acid

(0.1 mol/L) used to reduce the salivary pH to 4.0 and was

expressed as mL of lactic acid.

2.4. Salivary protein concentrations and salivary amylaseactivity

The saliva protein concentration was determined by Lowry

method.6 Briefly, four different solutions were used: (A) 2%

Na2CO3 in 0.1 M NaOH; (B) 0.5% CuSO4�5H2O and 1% sodium

citrate; (C) 50 mL of solution A and 1 mL solution B and (D)

Folin Ciocalteu diluted with deionized water. A standard

solution of 0.1% bovine serum albumin (BSA) in 1% NaOH, was

used to the calibration curve with eight different concentra-

tions of protein (5, 10, 20, 40, 50, 80, 100, 200 mg/mL). The

volume of saliva per sample used was 10 mL. To this volume,

190 mL of deionized water and 3 mL of solution C were added.

After 10 min, 300 mL of solution D was added to the samples

and agitated. After 30 min period, the absorbance readings

were done at 660 nm in a spectrophotometer (Hitachi U-1100

Spectrophotometer). Salivary amylase activity was quantified

by kinetic method at 405 nm, using 2-chloro-p-nitrophenyl-a-

D-maltotrioside (CNPG3) as a substrate (Kit Amilasa 405,

Wiener Lab. 2000, Rosario, Argentina), following the manu-

facturer’s instructions.

2.5. Salivary IgA concentration

Enzyme-linked immunosorbent assay (ELISA) was used to

determine the IgA salivary levels, modified from the standard

protocol used for measurement of IgA blood levels. IgA reacts

with a specific antibody (anti-serum anti-IgA, Wiener Lab.

2000, Rosario, Argentina) forming insoluble complexes. The

turbidity formed by these complexes is proportional to the

concentration of IgA in the sample and can be read at 340 nm

in spectrophotometer. The calibration curve was obtained

through calibrator proteins (Wiener Lab. 2000, Rosario,

Argentina) diluted in saline solution at 1:10, 1:20, 1:40, 1:80

and 1:160 concentrations. The absorbance was read before

(DO1) and after antiserum incubation for 30 min (DO2). The DA

was determined and IgA concentrations were expressed as mg/

mL of saliva.

2.6. Salivary concentration of fluoride [FS] and calcium[Ca++]

For the analysis of ionized calcium concentrations, 80 mL of

each saliva sample was used. To this sample, 16 mL of ionic

strength adjuster for calcium (model ISA-932011, Orion

Research Inc., MA, USA) was added and then the calcium

concentration ([Ca++]) was determined using a specific calcium

electrode (model 9320BN, Orion) and a reference microelec-

trode (Analyzer) connected to a previously calibrated ion

analyser (Orion 720A+). The analyses were expressed in mV

and carried out in duplicates. The calibration curve was made

with five different concentrations of calcium (10, 20, 40, 80 and

160 Ca++ mg/mL) obtained from the standard solution of Ca++

(model 922006A, Orion Research Inc.). Calcium ion concentra-

tion in the saliva of rats was calculated as Ca++ mg/mL of saliva.

Calcium concentration was expressed by SFR as Ca++ mg/min/

100 g.

Salivary fluoride concentrations ([F�]) were determined by

an ion-specific electrode (model 9409BN, Orion) and a

reference microelectrode (Analyzer) connected to an ion

analyser (Orion 720A+). The set was calibrated with standard

fluoride concentrations at 0.15, 0.3, 0.6, 1.2 and 2.4 F� mg/mL,

obtained by serial dilution, with pH adjustment solution

(TISAB II, Orion). The readings were taken in mV and in

duplicates. Fluoride ion concentration in the saliva of rats was

Table 1 – Weight and salivary biochemical parameters of normotensive (Wistar) and spontaneously hypertensive rats(SHRs) at different ages.

Analysis Groups

Wistar 4 weeks Wistar 12 weeks SHR 4 weeks SHR 12 weeks

Weight (g) 80.4 � 3.96£ 353.2 � 9.3c 45.0 � 4.1l 235.4 � 4.5g

pH 8.1 � 0.07 7.9 � 0.09 8.3 � 0.03 8.3 � 0.06*

Salivary buffering capacity (SBC) (citric acid mL/SFR) 2.84 � 0.4 1.43 � 0.35# 2.46 � 0.43 3.21 � 0.51

IgA concentration (mg/min/100 g) 42.7 � 25.8 46.7 � 2.02 56.7 � 14.8 46.8 � 14.02

The results were expressed as means � SEM (n = 8–10) and statistically analysed by ANOVA ( p < 0.05). In the first line, £, c, l, and g indicate

significant differences amongst groups (mean weight accompanied by £ differs statistically from c, l and g; c differs statistically from £, l and

g; l differs statistically from £, c and g; g differs statistically from £, c, l). In the second line, * indicates significant difference between SHR 12

weeks and Wistar 12 weeks. In the third line, # indicates significant difference between Wistar 12 weeks and the other groups.

a r c h i v e s o f o r a l b i o l o g y 5 7 ( 2 0 1 2 ) 1 3 2 0 – 1 3 2 61322

calculated as F� mg/mL of saliva, and it was expressed by SFR

as F� mg/min/100 g.

2.7. Statistical analysis

The data were expressed as means � standard error of the

mean (SEM) and analysed by two-way ANOVA and Tukey post

test. Some results were analysed by Student’s t test. Signifi-

cance level within groups (normotensive or SHR) or across all

groups was set at p < 0.05.

3. Results

Physiological parameters were compared between different

ages (4 and 12 weeks old) into the same group and between

Wistar and SHR groups in same age. At 12 weeks, SHR

presented higher SBP mean values (161 � 4 mmHg, n = 10)

than Wistar rats (110 � 4 mmHg, n = 10). The body weight of

the rats increased with growth in both groups (Table 1),

however throughout the study, when normotensive and

hypertensive rats in the same ages were compared, the SHR

body weights were significantly lower than those of the Wistar

rats. Since the body weight (bw) of SHR was reduced when

0.00

0.01

0.02

0.03

4weeks

12weeks

4weeks

12 weeks

Wistar SHR

* **

Saliv

ary

Flow

Rat

em

L/m

in/1

00g

Fig. 1 – Salivary flow rate (mL/min/100 g body weight) of

normotensive (Wistar, n = 10) and hypertensive (SHR,

n = 10) rats at different ages. Bars represent means W SEM

of data. *p = 0.0028, 4 and 12 weeks old Wistar;

**p = 0.0021, 12 weeks old Wistar and SHR (ANOVA and

Student’s t test).

compared to Wistar rats, the SFR was normalized to the weight

of the animals. Increased SFR (Fig. 1) was observed in 12-week-

old when compared to 4-week-old Wistar rats. Any alteration

on SFR was observed between 4 and 12 weeks SHR. SHR at 12-

week-old showed a reduced SFR than Wistar rat at same age.

A slight increase in saliva pH value in SHR 12 weeks old rats

was observed when compared to Wistar rat at same age (Table

1).

As body weight and SFR were reduced in SHR, all results of

biochemical analysis were normalized to the SFR based on

body weight.

A reduced SBC was observed only in 12-weeks-old Wistar

rats when compared to other groups (Table 1). The saliva IgA

concentration was not different between groups (Table 1).

Protein concentration in the saliva and specific amylase

activity were not altered by growth in Wistar group (Figs. 2

and 3). In SHR, the total protein concentration in saliva showed

a threefold increase in 12-week-olds when compared with 4-

week-olds (Fig. 2), associated to an increase of the specific

amylase activity (Fig. 3) in these animals.[Ca++] was increased

in the saliva of 12-week-old Wistar when compared to 4-week-

old rats (Fig. 4). A reduced [Ca++] was observed in SHR when

compared to Wistar at 12 weeks (Fig. 4). The [F�] was higher in

12 than 4 weeks old Wistar rats and in SHR group (Fig. 5).

0

50

100

150

200

250

300

350

400

***

Wistar SHR

4weeks

4weeks

12weeks

12weeks

Prot

ein

Con

cent

ratio

nμ

g/m

in/1

00g

Fig. 2 – Salivary protein concentration (mg/min/100 g body

weight) of normotensive (Wistar, n = 10) and hypertensive

(SHR, n = 10) rats at different ages. Bars represent

means W SEM of data. *p = 0.0198, 4 and 12 weeks old SHR;

**p = 0.0240, 12 weeks old Wistar and SHR (ANOVA and

Student’s t test).

0

25

50

75

100*

Wistar SHR

4weeks

4weeks

12weeks

12weeks

Am

ylas

eU

/min

/100

g

Fig. 3 – Amylase activity in the saliva of rats (n = 10)

expressed as U/min/100 g body weight. Bars represent

means W SEM of data. *p < 0.05 (ANOVA and Student’s t

test).

0.0

0.5

1.0

1.5

2.0

2.5

3.0

4weeks

12weeks

4weeks

12weeks

Wistar SHR

**

F-

(μg/

min

/100

g)

Fig. 5 – Fluoride ion concentration in the saliva of rats

(n = 10) was calculated as FS mg/mL of the saliva by SFR,

and expressed as FS mg/min/100 g. Bars represent

means W SEM of data. *p < 0.05 (ANOVA and Student’s t

test) between 4 and 12 weeks rats.

a r c h i v e s o f o r a l b i o l o g y 5 7 ( 2 0 1 2 ) 1 3 2 0 – 1 3 2 6 1323

The fluoride concentration in water and food was

0.068 ppm (mg F/L, average of samples) and 18.21 mg F/kg,

respectively. By assessing the amount of daily fluoride (mg F)

intake per body weight (kg) of each animal during 12 weeks, we

observed that Wistar and SHR ingested the same quantity of

fluoride (Wistar, 1.56 mg F/kg/day; SHR, 1.57 mg F/kg/day).

4. Discussion

In this study, the SHR was used as experimental model of

hypertension, since the haemodynamic characteristics of the

SHR are very similar to those of human essential arterial

hypertension. These animals are born normotensive with

average arterial pressure around 112 mmHg and develop

spontaneously an increase in arterial pressure from the 8th

0

500

1000

1500

4weeks

12weeks

12weeks

Wistar SHR

4weeks

* *

Ca++

( µg/

min

/100

g)

Fig. 4 – Calcium ion concentration in the saliva of rats

(n = 10) was calculated as Ca++ mg/mL of the saliva by SFR,

and expressed as Ca++ mg/min/100 g. Bars represent

means W SEM of data. *p < 0.05 (ANOVA and Student’s

t test).

week after birth7 reaching values higher than 150 mmHg at 12

weeks of age. It has been widely accepted that the most

appropriate control strain to SHR studies is the Wistar-Kyoto

(WKY) rat, to which SHR rats are genetically related. Concerns

have been raised about genetic differences8 and biological

variability9 between SHR and WKY rats. Moreover, evidence

suggest that the WKY strain is not the most suitable for

backcross studies because of the incidence of spontaneous

hypertension and the somewhat higher levels of blood

pressure in these rats.10–13 According to several studies,4,14,15

SHRs were compared to Wistar rats, which are safely

normotensive and with no genetic alteration that could

modulate arterial pressure.

In this study, SHR showed lower body weight compared to

the normotensive controls, regardless of the age evaluated.

These results suggest a growth delay of SHR. It could be

associated with the genotype of these animals. However,

different evidence raise the hypothesis that both pre and

postnatal periods are directly related to maternal contact and

contribute more significantly to the growth delay in SHR rather

than the genetic susceptibility.16–18 As previously ob-

served,2,4,7,19–22 the mean weight gain of female SHR during

pregnancy and lactation periods, SHR foetal weight, litter size

and postpartum development of SHR pups were lower than

those observed for normotensive rats. Maternal factors acting

in the uterus or through the milk would have major impact on

the pre and postnatal development of SHR. These factors seem

to be mainly correlated with the nutrition of the foetus or

newborn rat.16–18 Alterations in the mammary gland activity

were also observed in female SHR,23 with production of lower

quality and quantity of milk.

Clinical and experimental studies associate the reduction

of salivary activity with pre or postnatal delayed development,

resulted from deficient nutrition or related factors. Under-

nourished children have the stimulated SFR reduced.24

Nineteen-day-old Sprague-Dawley rats treated with a

a r c h i v e s o f o r a l b i o l o g y 5 7 ( 2 0 1 2 ) 1 3 2 0 – 1 3 2 61324

deficient protein diet had reduced body weight and SFR.25

Deprivation of iron in the diet also decreases the SFR in 21-day-

old rats, suggesting that lack of iron in this period of growth

and development causes changes in the salivary gland

activity.26

As observed in the present study, SHR at the different ages

showed reduced salivary parameters when compared with

their respective normotensive controls. We observed a

significant increase in the SFR of 12-week-old in relation to

4-week-old normotensive rats. This observation is in agree-

ment with other experimental and clinical studies that

associated the SFR increase with human and animal develop-

ment. Clinically, it has been demonstrated that the SFR

increases progressively from childhood to adolescence.27

However, this increase was not observed between SHR at

different ages.

We have previously observed3 that 4-week-old SHR had

reduced SFR stimulated by pilocarpine when compared with

Wistar rats of the same age. In the present study, reduced SFR

was noted when 12-week-old SHR was compared to Wistar rat

at same age. The salivary flow values (per animal weight) were

not different between 4 and 12-week-old SHR. Thus, these data

suggest that the altered SFR was maintained even with the

growth/development of these animals. Other authors28,29 also

observed reduced SFR after pilocarpine stimulation in 22-week-

old SHRs or after isoproterenol stimulation in 16–18-week-old

SHR, supposing that the SFR in SHR is reduced, regardless of the

type of stimulation (muscarinic or adrenergic).

All together, the results demonstrated that the reduced SFR

observed in SHR was independent of the age or the rise of

arterial blood pressure. However, evidence strongly suggest

that the genotype and deficient nutrition in pre and postnatal

periods could be directly associated to salivary hypofunction

observed in SHR.

Besides the reduced flow rate, the salivary hypofunction

has been characterized by alterations in the SBC, as well as in

the concentrations of organic and inorganic compounds

present in the saliva. We observed that the development of

normotensive rats was not associated with changes in salivary

pH but was associated with a decrease in SBC. The SBC is

measured by the activity of inorganic orthophosphate and

carbonic acid/bicarbonate system. Under conditions of sali-

vary flow stimulation, the bicarbonate buffer system repre-

sents 90% of the SBC. The concentration of bicarbonate in the

saliva depends on the SFR.30 We noticed an unaltered SBC in

12-week-old SHR regardless the reduced salivary flow rate of

these animals.The statistical data showed that the total

salivary protein concentration was not changed during the

growth/development of normotensive rats. Since the protein

concentration represents the amount of protein secreted by

the volume of saliva and the salivary flow increased during the

development of these animals, our results suggest that the

amount of protein secreted in the saliva of 12-week-old rats

was higher than that in 4-week-old Wistar rats. This

assumption could be reinforced by the unchanged amylase

activity detected in 12-week-old Wistar rats. On the other

hand, the concentration of protein secreted in the saliva was

almost threefold higher in 12 than in 4-week-old SHR, but the

SFR was not changed for these animals. Indeed, the increased

protein secretion was associated with the amylase activity

that was increased in the saliva of 12-week-old SHR.These

data might suggest that the growth/development or the

separation of pups from the mother prompted SHR to recover

the nutritional deficiency through diet. Indeed, the high

sympathetic activity detected in SHR31,32 might induce the

salivary protein secretion by b-adrenergic receptor activation.

Gradual increase of sympathetic stimulus was reported to be

parallel to the increase in salivary protein content also in

normal rats.33The lack of change in the saliva IgA concentra-

tion of normotensive and hypertensive rats at different ages,

despite the increased SFR observed in normotensive rats,

suggests that the secretion of immunoglobulins in saliva is not

modulated by age or hypertension. Probably, other factors like

autonomic stimulation, preganglionic parasympathectomy or

infectious systemic diseases34–37 could alter the saliva IgA

concentration in rats.

The salivary calcium comes from zymogen granules

secreted by acinar cells, releasing two types of calcium, free

and bound to proteins. In addition, calcium is actively

transported from the extracellular fluid by acinar cells and/

or ductal segment to the saliva. The salivary calcium

concentration is proportional to the plasmatic concentra-

tion,38 depending on the salivary flow and can also be

influenced by certain drugs such as pilocarpine, which could

increase calcium concentration. In our experiments, we

observed a significant correlation of the increase of salivary

calcium concentration, increased SFR and growth/develop-

ment of normotensive rats. However, this correlation could

not be accepted to SHR, since the calcium concentration and

the SFR were not altered between 4 and 12 weeks old SHR.

The presence of fluoride in the saliva is crucial for the tooth

mineral stability. The ability of saliva to maintain the fluoride

level constant in the tooth surface makes this fluoride source

an important element in the protection against caries by

promoting remineralization and reducing desmineraliza-

tion.39 In experimental models, the presence of fluoride in

the saliva depends on its absorption from exogenous sources.

Wistar rats and SHR were kept with their mothers until the 4th

week after birth and milk was their only source of food; so the

low concentration of fluoride in the saliva at 4 weeks old rats

would be directly proportional to the concentration of fluoride

present in the milk, or to the low milk intake during

breastfeeding. Concentrations of fluoride that account for

50% or less than the plasma concentration, were found in milk

of women, mares and cows.40 Our results showed that the

fluoride concentration in the saliva of Wistar rats and SHR at

12 weeks was significantly higher than that in the saliva of rats

at 4 weeks. In our study, the rats were fed with a standard diet

and water ad libitum after separation from the mothers (30

days after birth). These data reinforce the assumption that the

salivary fluoride concentration is proportional to the fluoride

content in the food. As the quantity of fluoride ingested is not

different between groups, these data pointed the absence of

fluoride pharmacokinetic alterations in SHR.

In conclusion, the present findings indicate that the

growth/development was associated to the increase of SFR

and to the increase of most biochemical parameters analysed

in normotensive rats. However, in SHR, the growth/develop-

ment did not alter the SFR, but age-related hypertension

modulated some parameters as salivary protein, amylase

a r c h i v e s o f o r a l b i o l o g y 5 7 ( 2 0 1 2 ) 1 3 2 0 – 1 3 2 6 1325

activity and fluoride concentration that were increased in 12

weeks SHR.

Funding

None.

Competing interest

None declared.

Ethical approval

All experiments in this study are in accordance with Ethical

Principles of Animal Experimentation (COBEA) and were

previously approved by Ethics Committee in Animal Experi-

mentation (ECAE), School of Dentistry of Aracatuba, UNESP,

according to the protocol 2007-003176.

Acknowledgements

This work was supported by the Foundation for Support

Research of the State of Sao Paulo (FAPESP-2007/50157-2),

National Council of Technological and Scientific Development

(CNPq), Brazilian Federal Agency for Support and Evaluation of

Graduated Education (CAPES) and UNESP Research Interna-

tionalization Program (PROINTER/PROPe – UNESP).

r e f e r e n c e s

1. Di Nicolantonio R, Koutsis K, Wlodek ME. Fetal versusmaternal determinants of reduced fetal and placentalgrowth in spontaneously hypertensive rat. Journal ofHypertension 2000;18(1):45–50.

2. Wlodek ME, Koutsis K, Westcott KT, Ho PW, Di NicolantonioR, Moseley JM. The spontaneously hypertensive rat fetus,not the mother, is responsible for the reduced amniotic fluidPTHrP concentrations and growth restriction. Placenta2001;22(7):646–51.

3. Elias GP, Dos Santos OA, Sassaki KT, Delbem AC, AntonialiC. Dental mineralization and salivary activity are reduced inoffspring of spontaneously hypertensive rats (SHR). Journalof Applied Oral Science 2006;14(4):253–9.

4. Elias GP, Sassaki KT, Delbem AC, Antoniali C. Atenololreduces salivary activity in pups of spontaneouslyhypertensive and normotensive rats treated duringpregnancy and lactation. Clinical and ExperimentalHypertension 2008;30(2):133–41.

5. Bernarde MA, Fabian FW, Rosen S, Hoppert CA, Hunt HR. Amethod for collection of large quantities of rat saliva. Journalof Dental Research 1956;35(2):326–7.

6. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Proteinmeasurement with the Folin phenol reagent. Journal ofBiological Chemistry 1951;193(1):265–75.

7. Yamori Y, Swales JD. The spontaneously hypertensive rat.In: Swales JD, editor. Textbook of hypertension. Oxford:Blackwell Scientific Press; 1994. p. 447–55.

8. Charchar FJ, Kaiser M, Bingham AJ, Fotinatos N, Ahmady F,Tomaszewski M, et al. Whole genome survey of copynumber variation in the spontaneously hypertensive rat:relationship to quantitative trait loci, gene expression, andblood pressure. Hypertension 2010;55(5):1231–8.

9. Kurtz TW, Morris Jr RC. Biological variability in Wistar-Kyoto rats. Implications for research with thespontaneously hypertensive rat. Hypertension1987;10(1):127–31.

10. Ashida T, Kuramochi M, Omae T. Increased sodium–calciumexchange in arterial smooth muscle of spontaneouslyhypertensive rats. Hypertension 1989;13(6 Pt 2):890–5.

11. Alexander D, Gardner JP, Tomonari H, Fine BP, Aviv A. LowerNa(+)–H+ antiport activity in vascular smooth muscle cellsof Wistar-Kyoto rats than spontaneously hypertensive andWistar rats. Journal of Hypertension 1990;8(9):867–71.

12. Louis WJ, Howes LG. Genealogy of the spontaneouslyhypertensive rat and Wistar-Kyoto rat strains: implicationsfor studies of inherited hypertension. Journal ofCardiovascular Pharmacology 1990;16(Suppl. 7):S1–5.

13. Sato T, Arai M, Goto S, Togari A. Effects of propranolol onbone metabolism in spontaneously hypertensive rats.Journal of Pharmacology and Experimental Therapeutics2010;334(1):99–105.

14. Bastos MF, Brilhante FV, Goncalves TED, Pires AG, NapimogaMH, Marque MR, et al. Hypertension may affect tooth-supporting alveolar bone quality: a study in rats. Journal ofPeriodontology 2010;81(7):1075–83.

15. Kristek F, Koprodova R, Cebova M. Long-term effects of earlyadministered Sildenafil and NO donor on the cardiovascularsystem of SHR. Journal of Physiology and Pharmacology2007;58(1):33–43.

16. McCarty R, Fields-Okotcha C. Timing of preweanlingmaternal effects on development of hypertension in SHRrats. Physiology and Behavior 1994;55(5):839–44.

17. Gouldsborough I, Black V, Johnson IT, Ashton N. Maternalnursing behaviour and the delivery of milk to the neonatalspontaneously hypertensive rat. Acta PhysiologicaScandinavica 1998;162(1):107–14.

18. Di Nicolantonio R, Koustsis K, Westcott KT, Wlodek ME.Relative contribution of the prenatal versus postnatal periodon development of hypertension and growth rate of thespontaneous hypertensive rat. Clinical and ExperimentalPharmacology and Physiology 2006;33(1–2):9–16.

19. Schenkels LC, Veerman EC, Nieuw Amerongen AV.Biochemical composition of human saliva in relation toother mucosal fluids. Critical Reviews in Oral Biology andMedicine 1995;6(2):161–75.

20. Lewis RM, Batchelor DC, Bassett NS, Johnston BM, Napier J,Skinner SJ. Perinatal growth disturbance in thespontaneously hypertensive rat. Pediatric Research1997;42(6):758–64.

21. Di Nicolantonio R, Koutsis K, Wlodek ME. Fetal versusmaternal determinants of the reduced fetal and placentalgrowth in spontaneously hypertensive rats. Journal ofHypertension 2000;18(1):45–50.

22. Wlodek ME, Westcott KT, Ho PW, Serruto A, Di NicolantonioR, Farrugia W, et al. Reduced fetal, placental, and amnioticfluid PTHrP in the growth-restricted spontaneouslyhypertensive rat. American Journal of Physiology RegulatoryIntegrative and Comparative Physiology 2000;279(1):R31–8.

23. Wlodek ME, Westcott KT, Serruto A, O’Dowd R, Wassef L, HoPW, et al. Impaired mammary function and parathyroidhormone-related protein during lactation in growth-restricted spontaneously hypertensive rats. Journal ofEndocrinology 2003;178(2):233–45.

24. Psoter WJ, Reid BC, Katz RV. Malnutrition and dental caries:a review of the literature. Caries Research 2005;39(6):441–7.

a r c h i v e s o f o r a l b i o l o g y 5 7 ( 2 0 1 2 ) 1 3 2 0 – 1 3 2 61326

25. Ryberg M, Johansson I. The effects of long-term treatmentwith salmeterol and salbutamol on the flow rate andcomposition of whole saliva in the rat. Archives of OralBiology 1995;40(3):187–91.

26. Enberg N, Alho H, Loimaranta V, Lenander-Lumikari M.Salivary flow rate, amylase activity, and protein andelectrolyte concentrations in saliva after acute alcoholconsumption. Oral Surgery Oral Medicine Oral Pathology OralRadiology and Endodontics 2001;92(3):292–8.

27. Roth GI, Calmes RB. Salivary glands and saliva. In: Roth GI,Calmes RB, editors. Oral biology. St. Louis, MO: CV Mosby Co.;1981. p. 196–236.

28. Kraly FS, Coogan LA, Specht SM, Trattner MS, Zayfert C,Cohen A, et al. Disordered drinking in developingspontaneously hypertensive rats. American Journal ofPhysiology 1985;248(4 Pt 2):R464–770.

29. Schmid G, Geiger H, Bahner U, Heidland A. Glandularadenylate cyclase system in genetic hypertension: age-dependent response to catecholamines. European Journal ofPharmacology 1988;147(3):397–402.

30. Bardow A, Nyvad B, Nauntofte B. Relationships betweenmedication intake, complaints of dry mouth, salivary flowrate and composition, and the rate of toothdemineralization in situ. Archives of Oral Biology2001;46(5):413–23.

31. Hojna S, Kunes J, Zicha J. Alterations of NO synthaseisoforms in brain and kidney of rats with genetic and salthypertension. Physiological Research 2010;59(6):997–1009.

32. Kishi T, Hirooka Y, Konno S, Ogawa K, Sunagawa K.Angiotensin II type 1 receptor-activated caspase-3 throughras/mitogen signal-regulated kinase in the rostralventrolateral medulla is involved in sympathoexcitation in

stroke-prone spontaneously hypertensive rats. Hypertension2010;55(2):291–7.

33. Anderson LC, Garret JR, Zhang X, Proctor GB, Shori DK.Differential secretion of proteins by rat submandibularacini and granular ducts on graded autonomicnerve stimulations. Journal of Physiology 1995;485:503–11.

34. Nogueira RD, King WF, Gunda G, Culshaw S, Taubman MA,Mattos-Graner RO, et al. Mutans streptococcal infectioninduces salivary antibody to virulence proteins andassociated functional domains. Infection and Immunity2008;76(8):3606–13.

35. Langley R, Wines B, Willoughby N, Basu I, Proft T, Fraser JD.The staphylococcal superantigen-like protein 7 binds IgAand complement C5 and inhibits IgA-Fc alpha RI bindingand serum killing of bacteria. Journal of Immunology2005;174(5):2926–33.

36. Carpenter GH, Proctor GB, Garrett JR. Preganglionicparasympathectomy decreases salivary SIgA secretion ratesfrom the rat submandibular gland. Journal ofNeuroimmunology 2005;160(1–2):4–11.

37. Carpenter GH, Proctor GB, Ebersole LE, Garrett JR. Secretionof IgA by rat parotid and submandibular cells in response toautonomimetic stimulation in vitro. InternationalImmunopharmacology 2004;4(8):1005–14.

38. Glijer B, Peterfy C, Tenenhouse A. The effect of vitamin Ddeficiency on secretion of saliva by rat parotid gland in vivo.Journal of Physiology 1985;363:323–34.

39. Dowd FJ. Saliva and dental caries. Dental Clinics of NorthAmerica 1999;43(4):579–97.

40. Whitford GM. The metabolism and toxicity of fluoride.Monographs in Oral Science 1989;13:1–160.