Low dose propranolol decreases orthodonticmovement

Erika Lira de Oliveira a, Fabiana Furtado Freitas b, Cristina Gomes de Macedo b,Juliana Trindade Clemente-Napimoga b, Milena Bortolotto Felippe Silva c,Luiz Roberto Coutinho Manha es-Jr c, Jose Luiz Cintra Junqueira c,Marcelo Henrique Napimoga a,*a Laboratory of Immunology and Molecular Biology, Sao Leopoldo Mandic Dental School and Research Center,

Campinas/SP, Brazilb Laboratory of Orofacial Pain, Department of Physiology, Piracicaba Dental School, State University of Campinas,

Piracicaba/SP, Brazilc Laboratory of Oral Radiology, Sao Leopoldo Mandic Dental School and Research Center, Campinas/SP, Brazil

a r c h i v e s o f o r a l b i o l o g y 5 9 ( 2 0 1 4 ) 1 0 9 4 – 1 1 0 0

a r t i c l e i n f o

Article history:

Accepted 18 June 2014

Keywords:

Propranolol

Tooth movement

Orthodontic

Bone

a b s t r a c t

Objective: Low dose propranolol has previously been demonstrated to suppress bone remo-

delling. Therefore, its effect on orthodontic movement was tested.

Design: Rats were assigned as follows (n = 5): animals with no orthodontic appliance (G1);

the remaining groups were fitted with a Ni-Ti closed-coil spring ligated to the upper left first

molar and connected to the incisors using metal and resin and received vehicle only (G2),

0.1 mg/kg (G3) or 20 mg/kg (G4) of propranolol orally. Cone Beam Computed Tomography

was performed using high resolution for image capture. The distance between the first and

second upper molars, both with and without the orthodontic appliance, was measured in

millimetres. Gingival tissue was harvested and assessed for IL-1b and IL-6 using ELISA and

for ICAM-1 and RANKL by Western blotting.

Results: The orthodontic appliance induced a significant tooth movement in G2 when

compared to the animals without an orthodontic appliance (G1) ( p < 0.05). The animals

from G3 showed a significantly reduction in tooth movement ( p < 0.05) when compared

with rats from G2. Animals treated with 20 mg/kg of propranolol (G4) showed tooth

movement similar to that of G2. The reduced tooth movement observed in the animals

treated with 0.1 mg/kg of propranolol (G3) occurred due to decreased amounts of IL-1b and

IL-6, in addition to lower ICAM-1 and RANKL expression.

Conclusions: Low dose propranolol inhibits bone remodelling and orthodontic movement.

# 2014 Elsevier Ltd. All rights reserved.

* Corresponding author at: Laboratory of Immunology and Molecular Biology, Sao Leopoldo Mandic Dental School and Research Center R.Jose Rocha Junqueira 13, Campinas, Sao Paulo 13045-755, Brazil. Tel.: +55 19 3211 3627; fax: +55 19 3211 3636.

E-mail addresses: napimogamh@yahoo.com, marcelo.napimoga@gmail.com (M.H. Napimoga).

Available online at www.sciencedirect.com

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

http://dx.doi.org/10.1016/j.archoralbio.2014.06.0060003–9969/# 2014 Elsevier Ltd. All rights reserved.

a r c h i v e s o f o r a l b i o l o g y 5 9 ( 2 0 1 4 ) 1 0 9 4 – 1 1 0 0 1095

Fig. 1 – Photo of the orthodontic appliance placement used

in the current study.

1. Introduction

During orthodontic movement, force is applied to the teeth,

and areas of pressure and tension are formed in the

periodontal ligament (PDL), the connective tissue that con-

nects the tooth to its surrounding alveolar bone.1 Mechanical

loading also alters periodontal tissue vascularity and blood

flow, which results in the synthesis and release of various

molecules, locally, such as neurotransmitters, cytokines,

growth factors, and arachidonic acid metabolites. These

molecules evoke a cellular response in the different cell types

surrounding the teeth, providing a favourable microenviron-

ment for bone deposition and resorption.2

Molecules present in drugs that are regularly consumed

by patients reach the mechanically stressed paradental

tissue via the circulation and interact with local target cells.

The combined effect of the mechanical forces and one or

more of these agents can be inhibitory, additive, or

synergistic3. The regulation of bone metabolism by the

sympathetic nervous system has been demonstrated in

studies showing that osteoblasts and osteoclasts express b2-

adrenoceptors.4,5 It has been previously demonstrated that

low doses of the b-blocker propranolol suppress alveolar

bone resorption by inhibiting RANKL-mediated osteoclasto-

genesis and inflammatory markers, with no affect on

haemodynamic parameters.6

b-Blockers have been classified as a first-line drug in the

treatment of hypertension and are widely used in cardio-

vascular disease. Globally, the prevalence of obesity has

been steadily increasing over recent decades. Hypertension

is commonly present in the overweight and obese popula-

tions. The US leads the developed world in terms of obesity

rates, with a prevalence of 34% and 17% in adults and

children aged 2–19 years, respectively.7 Projections based on

the current obesity trends predict that by 2030 there will be

another 65 million obese adults in the US,8 which will

consequently increase hypertension rates. Propranolol is

used in hypertension, angina, and migraine, amongst other

conditions. Considering that the number of hypertensive

patients is increasing, including in young people who are

the main target of orthodontic treatment, it is hypothesized

that a common b-blocker (propranolol) may influence

bone remodelling during orthodontic movement. The

objective of the present study was to investigate the effects

of low and high doses of propranolol on tooth movement

in rats.

2. Materials and methods

2.1. Animals

Three-month old male Wistar rats (200–250 g) were used. The

animals were kept in appropriate cages in a temperature-

controlled room under a 12-h dark/light cycle. Free access to

water and food was provided and they were acclimatized over

a period of approximately 7 days in the laboratory prior to the

experiment. All animals were manipulated in accordance with

the Guiding Principles in The Care and Use of Animals,

approved by the Council of the American Physiologic Society.

The Animal Ethics Committee of Sao Leopoldo Mandic Dental

School approved this study (# 2012/0287).

2.2. Appliance placement and measurement of toothmovement

The appliance placement was adapted from Gameiro et al.9

The animals were anaesthetized using xylazine (10 mg/kg)

and maintained with ketamine (50 mg/kg). A closed coil

nickel-titanium (NiTi) spring (Morelli1, Campinas, Brazil),

calibrated to provide a force of 0.49 N, measured by a

tensiometer (Morelli1, Campinas, Brazil) was ligated to the

upper left first molar and connected to incisors by

orthodontic stainless steel wire (.00800) (Morelli1, Campinas,

Brazil) and light-cured resin (Transbond XT, 3M1, Campinas,

Brazil) as illustrated in Fig. 1. NiTi springs were used to

provide a relatively constant force over the course of the

experiment. To limit the influence of inter-animal variation

a split-mouth design was used and the contralateral

untreated side served as the intra-animal control. After 10

days of tooth movement the rats were decapitated, and the

maxillae excised.

After the orthodontic appliance was positioned, the

animals were randomly assigned to one of the following

groups: 1) sham-appliance animals receiving administration

of saline (vehicle control) (n = 5); 2) animals with orthodontic

appliance receiving administration of vehicle (n = 5); 3)

animals with orthodontic appliance receiving administra-

tion of oral propranolol (0.1 mg/kg/day) (n = 5); 4) animals

with orthodontic appliance receiving administration of

oral propranolol (20 mg/kg/day) (n = 5). Saline (vehicle)

or propranolol was orally administered daily to each

animal.

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2.3. Measurement of tooth movement

After scout acquisition and protocol selection, Cone Beam

Computed Tomography (CBCT) equipment was calibrated

according to the manufacturer’s instructions to avoid humid-

ity and temperature variations, therefore maintaining image

quality. Classic I-Cat (Imaging Science International, Hatfield,

USA), model 914040000-0000R, was used to acquire images of

the maxillae of the rats. Voxel with a 0.25 mm � 6.00 cm field

of view and an exposure time of 40 s was selected for all

images captured. The X-ray settings were established using

equipment at 120 kV and 5–7 mA in accordance with the

resolution. The maxillae of the rats were positioned with the

occlusal plane directed upwards and held in place by wax. All

images were processed using XoranCatTM software (Xoran

Technologies Inc, Ann Arbor, USA). All anatomical plane slice

corrections and measurements were performed. In addition,

the Angio-Sharpen-Medium 5 � 500 filter was applied to all

images, and the contrast and brightness were adjusted in

order to give a more detailed image.

In the Multiplanar Reconstruction (MPR) images, the plane

correction arrows were used to construct the occlusal plane of

the maxillae of the rats in tangent to the coronal, sagittal and

axial planes. After correction, MPR plane cross-sections were

performed using the oblique tool (Fig. 2A), whilst the ruler tool

was used for measurements. The linear distance was

measured in the antero-posterior direction on the treated

side only and performed between the distal surface of the first

molar to the mesial surface of the second molar (Fig. 2B).

2.4. Protein extraction from gingival tissue

The marginal gingival tissue around the maxillary first molars

was surgically harvested (approximately 100 mg), rinsed with

cold sterile saline solution (0.9%) and triturated and homoge-

nized in 300 ml of the appropriate protease inhibitor-contain-

ing buffer (RIPA Lysis and Extraction Buffer, Thermo Scientific,

Fig. 2 – CBCT image used for measuring orthodontic movement.

and the second molars.

Rockford, IL, USA) and then centrifuged for 10 min at

10,000 � g. The total extracted protein was colorimetrically

measured using the micro BCA protein assay kit (Thermo

Scientific). The supernatant was stored at �70 8C until further

analysis. In addition, the maxillae were removed and fixed in

4% neutral formalin for 48 h for Cone Beam Computed

Tomography image acquisition, in order to determine the

amount of tooth movement.

2.5. Enzyme-linked immunosorbent assay (ELISA)

The levels of IL-1b and IL-6 were determined via capture

enzyme-linked immunosorbent assays (ELISA) using protocols

supplied by the manufacturer (R&D Systems Minneapolis,

USA). Fifty ml of gingival homogenate samples were applied in

duplicate, and the plates incubated for 2 h at room tempera-

ture. All experiments included serial dilutions (800, 400, 200,

100, 50, 25, 12.5 and 6.25 pg/ml) of a standard sample of mouse

IL-1b or IL-6 protein. The secondary antibody was biotin-

conjugated at a dilution of 1:1000. After incubation with a

solution of avidin–peroxidase for 30 min at room temperature,

a further series of washes was performed and 100 ml of

3,30,5,50-Tetramethylbenzidine substrate (TMB) was added and

incubated for 15 min. Absorbance values (A450 nm) were

obtained using an ELISA plate reader (Microplate Reader/

Model 3550, Bio Rad). Negative controls did not include

gingival homogenate. Absorbance values were plotted against

the standard curve obtained for the serial dilutions of the

purified mouse standard within a linear range to determine IL-

1b and IL-6 concentrations. ELISA was carried out in a blind

fashion.

2.6. Western blotting

Equal amounts of protein (20 mg) from the gingival tissue of the

animals were separated using 10% sodium dodecyl sulfate–

polyacrylamide gel electrophoresis and transferred to a

(A) Cross-section image; (B) linear distance between the first

Fig. 3 – Orthodontic tooth movement. The tooth movement

following orthodontic pressure was measured using the

XoranCatTM software. The distance is presented in

millimetres. Data are representative of 3 independent

experiments in duplicate (n = 5 per group). Different letters

indicate statistical significance (One Way ANOVA followed

by Bonferroni test).

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nitrocellulose membrane (Bio-Rad Laboratories). A molecular

weight standard (Bio-Rad Laboratories) was run in parallel to

estimate molecular weight. Membranes were blocked over-

night at 4 8C in Tris-buffered saline-Tween (20 mM Tris–HCl,

pH 7.5, 500 mM NaCl, 0.1% Tween 20; TBST) containing 5%

dried milk. The membranes were then incubated at 4 8C

overnight with anti-RANKL (Receptor activator of nuclear

factor kappa-B ligand; 1:1000), anti-ICAM-1 (Intercellular

Adhesion Molecule-1; 1:2000) or a-tubulin (1:1000) (Santa Cruz

Biotechnology, Santa Cruz, CA, USA), and diluted in TBST

containing 5% dried milk. Membranes were then incubated at

room temperature for 60 min with a secondary antibody

conjugated with peroxidase (1:5000), also diluted in TBS-T

containing 5% dried milk. The bands recognized by the specific

antibody were visualized using a chemiluminescence-based

ECL system (Amersham Biosciences, Piscataway, NJ) and

exposed to an X-ray film for 30 min (Eastman Kodak,

Rochester, NY). A computer-based imaging system (Image J)

was used to measure the intensity of optical density of the

bands.

2.7. Statistical analyses

Statistical analysis was performed using the GraphPad Prism

4.0 software (La Jolla, CA, USA). Data were reported as

means � SD, with five animals per group. The means from

different treatments were compared using ANOVA. When a

significant difference was identified, individual comparisons

were subsequently performed using Bonferroni’s t-test for

unpaired values. Statistical significance was set at p < 0.05.

3. Results

3.1. Low dose propranolol decreases orthodonticmovement

All animals gained weight during the study, however, the

mean body weight was not significantly different between the

Fig. 4 – Effects of propranolol in different doses on cytokines ex

gingival tissue around the teeth orthodontically moved. Results

group). Different letters indicate statistical significance (One Wa

groups at the end of the experimental period (data not shown).

The orthodontic appliance significantly increased tooth

movement (4.1-fold) compared to the control group (Fig. 3;

p < 0.05). Oral administration of low dose propranolol (0.1 mg/

kg) markedly reduced orthodontic movement in 41% (Fig. 3;

p < 0.05) when compared to orthodontic movement in animals

receiving the vehicle. On the other hand, high dose proprano-

lol (Fig. 3; 20 mg/kg) did not significantly reduce orthodontic

movement (11%).

3.2. Cytokine measurements from gingival tissue

IL-1b and IL-6 are critical cytokines in bone biology, therefore,

the levels of both cytokines present in the gingival tissue

pression. Cytokine quantification was performed using the

are expressed as means (pg/mg of tissue) WSD (n = 5 per

y ANOVA followed by Bonferroni test).

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subjected to the orthodontic appliance following 10 days of

propranolol treatment was evaluated. Expression of both IL-1b

and IL-6 was significantly increased in the group using the

orthodontic appliance, when compared to the sham-appliance

group. Interestingly, only the animals treated with 0.1 mg/kg

of propranolol demonstrated a significant decrease in expres-

sion of both cytokines ( p < 0.05) (Fig. 4A and B, respectively).

3.3. RANKL and ICAM-1 expression

The expression of two important molecules was analyzed using

Western blotting: RANKL, a key molecule in the activation of

osteoclasts; and ICAM-1, an endothelial- and leucocyte-associ-

ated transmembrane protein. As shown in Fig. 5, RANKL was

upregulated ( p < 0.05) in the gingival tissue of the molar teeth

subjected to orthodontic forces, when compared to the control

group. In contrast, the animals that received 0.1 mg/kg showed

a significantly decreased expression ( p < 0.05), whilst 20 mg/kg

of propranolol showed no difference in expression of this

molecule. ICAM-1 expression was also raised in the gingival

tissue of the molars only subjected to orthodontic forces

( p < 0.05), whilst the animals treated with 0.1 mg/kg of

propranolol in addition to orthodontic forces had statistically

Fig. 5 – Effects of propranolol on RANKL expression during

orthodontic tooth movement. Protein expression was

analyzed via Western blotting. The intensity of the bands

in terms of optical density was measured and normalized

against a-tubulin expression. Protein band intensity is

represented as arbitrary units. The results are expressed

as mean W SD of five animals per group. Different letters

indicate statistical significance (One Way ANOVA followed

by Bonferroni test).

decreased levels of ICAM-1 expression ( p < 0.05). On the other

hand, animals subjected to orthodontic forces during treatment

with 20 mg/kg of propranolol showed the same ICAM-1

expression as demonstrated in the aforementioned group, for

which only an orthodontic appliance was used (Fig. 6).

4. Discussion

Studies have indicated that b2-adrenergic receptors mediate

signalling in osteoblasts, which inhibits bone formation and

increases osteoclastogenesis via receptor activation of nuclear

factor kappa-B ligand (RANKL) expression.10,11 Kondo et al.12

reported that bone loss induced by mechanical unloading is

regulated by the sympathetic nervous system. The present

study corroborate their results by demonstrating that block-

ade of sympathetic signalling with low dose propranolol

inhibits orthodontic movement by reducing important mole-

cules responsible for bone remodelling.

It has been demonstrated that sympathetic signalling via

osteoclast activation controls the mechano-adaptive response

induced by experimental tooth movement.13 In the present

study, the use of low dose propranolol, a b-adrenergic

antagonist, decreased orthodontic movement. This may be

explained by the fact that osteoblasts and osteoclasts are well

equipped with adrenergic and peptidergic receptors, indicating

Fig. 6 – Effects of propranolol on ICAM-1 expression during

orthodontic tooth movement. Protein expression was

analyzed via Western blotting. The intensity of the bands

in terms of optical density was measured and normalized

against a-tubulin expression. Protein band intensity is

represented as arbitrary units. The results are expressed

as mean W SD of five animals per group. Different letters

indicate statistical significance (One Way ANOVA followed

by Bonferroni test).

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that they are influenced by sympathetic neurotransmitters.14

Additionally, osteoclast number, surface, and activity are

increased after sympathectomy, whereas osteoblastic activity

is decreased.15–18 Rodrigues et al. demonstrated that at low

doses, propranolol can inhibit bone resorption in a periodonti-

tis-induced rat model via inhibition of osteoclast differentiation

and resorptive activity due to suppression of the nuclear factor

of activated T cells (NFATc)1 pathway and the expression of

tartrate-resistant acid phosphatase (TRAP), cathepsin K and

MMP-9.6 It is important to highlight that the effect observed

using low dose propranolol did not evoke a haemodynamic

effect in the animals.

The present study demonstrated that only low dose

propranolol was able to decrease orthodontic tooth move-

ment. This data is in accordance with the literature, which

demonstrated that b1- and b2-adrenergic signalling exerts

opposing effects on bone, with b1 being predominantly an

anabolic stimulus in response to mechanical stimulation and

during growth, and b2 mainly regulating bone resorption.

Interestingly, mice lacking the adrenoreceptor b-2 (Adrb2R)

present a high bone mass phenotype, whilst Adrb 1 and 2R

deficient mice have reduced trabecular and cortical bone

mass. This suggests that high dose propranolol may some-

what imitate the double deletion phenotype,19 whilst low dose

propranolol may act mainly via the Adrb 2R, therefore exerting

beneficial effects.6 Hence, in the present study, low dose

propanolol may have blocked signalling by Adrb 2R and

consequently inhibited orthodontic movement, whereas high

doses may have blocked both Adbr 1 and 2 and therefore

prevented this effect.

Mechanical forces during orthodontic treatment can cause

an increased production of different cytokines by the

periodontal ligament cells, including IL-1b and IL-6. Interleu-

kin-1b is known to be responsible for neutrophil recruitment, a

complex process involving a sequence of molecular-mechani-

cal events on leukocytes and endothelial cells that depend on

distinct cell-cell adhesion molecules, such as ICAM-1, and

increased cytokines/chemokines production.20 Additionally,

IL-1b attracts macrophages and supports their differentiation

into osteoclasts, which carry out bone resorption. They also

inhibit the activity of osteoblasts, thus preventing bone

formation.21 This guarantees that necrotic tissue is resorbed

due to the initial application of force, and that tooth

movement occurs with bone remodelling. Therefore, the

inhibitory effect of low dose propranolol on IL-1b production

and ICAM-1 expression, explained in part the diminished

orthodontic movement observed in the present study.

Interleukin-6 regulates immune responses at sites of

inflammation, as well as an autocrine/paracrine activity that

stimulates osteoclast formation and bone-resorbing activity.22

It plays an important role in local regulation of bone remodelling

and is produced at the beginning of orthodontic tooth

movement (until 12 days after orthodontic activation), and its

expression decreases over time. A physiological homeostasis is

probably reached through downregulation via a feedback

mechanism.23 Furthermore, it is currently known that IL-6

can upregulate RANKL, indirectly supporting osteoclast forma-

tion via the interaction with mesenchymal cells. The cytokine

RANKL is essential for osteoclast development in bone and is

abundantly found at the pressure site during orthodontic

movement.24 Here, we have demonstrated that low dose of the

betablocker propranolol decreases the amount of both mole-

cules, IL-6 and RANKL, which further contributes towards the

understanding of how this drug inhibits orthodontic move-

ment. Likewise, previous data have shown that fenoterol (b2-

agonist) stimulated RANKL mRNA expression by nearly twofold

and this was suppressed by propranolol (b-blocker), suggesting

that b-adrenoceptors may play a role in modulating bone

turnover via the sympathetic nervous system.25

Based on the aforementioned results, it is possible to

conclude that low dose propranolol, a b-adrenergic antago-

nist, decreases orthodontic movement.

Funding

There is no governmental or private funding for this research.

Competing interest

There is no conflict of interest to declare.

Ethical approval

This study was approved by the Animal Ethics Committee of

the Faculty Sao Leopoldo Mandic (# 2012/0287).

r e f e r e n c e s

1. Alves JB, Ferreira CL, Martins AF, Silva GA, Alves GD, PaulinoTP, et al. Local delivery of EGF-liposome mediated bonemodeling in orthodontic tooth movement by increasingRANKL expression. Life Sci 2009;85(19-20):693–9.

2. Krishnan V, Davidovitch Z. Cellular, molecular, and tissue-level reactions to orthodontic force. Am J Orthod DentofacialOrthop 2006;129(4):469.e1–e.

3. Diravidamani K, Sivalingam SK, Agarwal V. Drugsinfluencing orthodontic tooth movement: an overall review.J Pharm Bioallied Sci 2012;4(Suppl. 2):S299–303.

4. Takeda S, Elefteriou F, Levasseur R, Liu X, Zhao L, Parker KL,et al. Leptin regulates bone formation via the sympatheticnervous system. Cell 2002;111(3):305–17.

5. Bonnet N, Benhamou CL, Malaval L, Goncalves C, Vico L,Eder V, et al. Low dose beta-blocker prevents ovariectomy-induced bone loss in rats without affecting heart functions. JCell Physiol 2008;217(3):819–27.

6. Rodrigues WF, Madeira MF, da Silva TA, Clemente-Napimoga JT, Miguel CB, Dias-da-Silva VJ, et al. Low dose ofpropranolol down-modulates bone resorption by inhibitinginflammation and osteoclast differentiation. Br J Pharmacol2012;165(7):2140–51.

7. Flegal KM, Carroll MD, Ogden CL, Curtin LR. Prevalence andtrends in obesity among US adults, 1999–2008. JAMA2010;303(3):235–41.

8. Wang YC, McPherson K, Marsh T, Gortmaker SL, Brown M.Health and economic burden of the projected obesity trendsin the USA and the UK. Lancet 2011;378(9793):815–25.

9. Gameiro GH, Nouer DF, Pereira-Neto JS, Urtado MB, NovaesPD, de Castro M, et al. The effects of systemic stress onorthodontic tooth movement. Aust Orthod J 2008;24(2):121–8.

a r c h i v e s o f o r a l b i o l o g y 5 9 ( 2 0 1 4 ) 1 0 9 4 – 1 1 0 01100

10. Takeuchi T, Tsuboi T, Arai M, Togari A. Adrenergicstimulation of osteoclastogenesis mediated by expression ofosteoclast differentiation factor in MC3T3-E1 osteoblast-likecells. Biochem Pharmacol 2001;61(5):579–86.

11. Elefteriou F, Ahn JD, Takeda S, Starbuck M, Yang X, Liu X,et al. Leptin regulation of bone resorption by thesympathetic nervous system and CART. Nature2005;434(7032):514–20.

12. Kondo H, Nifuji A, Takeda S, Ezura Y, Rittling SR, DenhardtDT, et al. Unloading induces osteoblastic cell suppressionand osteoclastic cell activation to lead to bone loss viasympathetic nervous system. J Biol Chem2005;280(34):30192–200.

13. Kondo M, Kondo H, Miyazawa K, Goto S, Togari A.Experimental tooth movement-induced osteoclastactivation is regulated by sympathetic signaling. Bone2013;52(1):39–47.

14. Togari A. Adrenergic regulation of bone metabolism:possible involvement of sympathetic innervation ofosteoblastic and osteoclastic cells. Microsc Res Tech2002;58(2):77–84.

15. Sandhu HS, Kwong-Hing A, Herskovits MS, Singh IJ. Theearly effects of surgical sympathectomy on boneresorption in the rat incisor socket. Arch Oral Biol1990;35(12):1003–7.

16. Hill EL, Turner R, Elde R. Effects of neonatal sympathectomyand capsaicin treatment on bone remodeling in rats.Neuroscience 1991;44(3):747–55.

17. Sherman BE, Chole RA. In vivo effects of surgicalsympathectomy on intramembranous bone resorption. Am JOtol 1996;17(2):343–6.

18. Sherman BE, Chole RA. Effect of pharmacologicalsympathectomy on osteoclastic activity in the gerbillineauditory bulla in vivo. Ann Otol Rhinol Laryngol 1999;108(11 Pt1):1078–87.

19. Pierroz DD, Bonnet N, Bianchi EN, Bouxsein ML, Baldock PA,Rizzoli R, Ferrari SL. Deletion of b-adrenergic receptor 1, 2,or both leads to different bone phenotypes and response tomechanical stimulation. J Bone Miner Res 2012;27(June(6)):1252–62.

20. Hogg JC, Walker BA. Polymorphonuclear leucocyte traffic inlung inflammation. Thorax 1995;50(8):819–20.

21. Nakamura K, Sahara N, Deguchi T. Temporal changes in thedistribution and number of macrophage-lineage cells in theperiodontal membrane of the rat molar in response toexperimental tooth movement. Arch Oral Biol 2001;46(7):593–607.

22. Uematsu S, Mogi M, Deguchi T. Interleukin (IL)-1 beta, IL-6,tumor necrosis factor-alpha, epidermal growth factor, andbeta 2-microglobulin levels are elevated in gingivalcrevicular fluid during human orthodontic tooth movement.J Dent Res 1996;75(1):562–7.

23. Madureira DF, Taddei Sde A, Abreu MH, Pretti H, Lages EM,da Silva TA. Kinetics of interleukin-6 and chemokine ligands2 and 3 expression of periodontal tissues during orthodontictooth movement. Am J Orthod Dentofacial Orthop2012;142(4):494–500.

24. Yamaguchi M. RANK/RANKL/OPG during orthodontic toothmovement. Orthod Craniofac Res 2009;12(2):113–9.

25. Huang HH, Brennan TC, Muir MM, Mason RS. Functionalalpha1- and beta2-adrenergic receptors in humanosteoblasts. J Cell Physiol 2009;220(1):267–75.