In vivo stimulation of sympathetic nervous system modulates osteoblastic activity in mouse calvaria
Ayami Kondo and
Akifumi Togari
Department of Pharmacology, School of Dentistry, Aichi-Gakuin University,
Nagoya 464-8650, Japan
Submitted 21 January 2003
; accepted in final form 25 April 2003
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ABSTRACT
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Previously, we demonstrated that epinephrine induced the expression of
interleukin (IL)-6 mRNA via
-adrenoceptors in cultured human
osteoblastic cells. IL-6 is well known to modulate bone metabolism by
regulating the development and function of osteoclasts and osteoblasts.
Recently, restraint stress and intracerebroventricular injection of
lipopolysaccharide (LPS) have been reported to induce the expression of IL-6
mRNA in peripheral organs in mice in which expression is mediated by the
activation of the sympathetic nervous system. To prove the physiological role
of sympathetic nerves in bone metabolism in vivo, we examined by RT-PCR
analysis the effects of restraint stress and intracerebroventricular injection
of LPS on IL-6 mRNA expression in mouse calvaria. The expression of IL-6 mRNA
in mouse calvaria was stimulated by either restraint stress (30 min) or
intracerebroventricular injection of LPS (50 ng/mouse, 60 min). The treatment
of mice with the neurotoxin 6-hydroxydopamine (6-OHDA, 100 mg ·
kg-1 · day-1 ip for 3 days)
inhibited LPS (icv)-induced expression of IL-6 mRNA in their calvaria. The
expression of IL-6 mRNA induced by the restraint stress was not influenced by
6-OHDA, which destroys noradrenergic nerve terminals. Furthermore,
pretreatment with a
-blocker, propranolol (15 or 25 mg/kg ip), inhibited
both stress- and LPS-induced increases in the level of IL-6 mRNA, but
pretreatment with an
-blocker, phentolamine (5 mg/kg sc), did not
inhibit them in mouse calvaria. In addition, treatment of calvaria with
isoprenaline or norepinephrine increased IL-6 synthesis in the organ culture
system. These results indicate that in vivo adrenergic stimulation modulates
the osteoblastic activity in mouse calvaria via noradrenergic nerve
terminals.
restraint stress; intracerebroventricular injection; interleukin-6; lipopolysaccharide; calvaria; sympathetic activity; osteoblast
HISTOCHEMICAL AND PHARMACOLOGICAL STUDIES indicate the
involvement of neural regulation in bone metabolism mediated by osteoblastic
and osteoclastic cells. Mammalian bones are widely innervated by sympathetic
and sensory nerves, which are particularly abundant in regions of high
osteogenic activity, such as the growth plate
(4,
15). In heterotropic bone
formation induced by demineralized bone matrix, an early in-growth of
noradrenergic nerves has been detected
(5). Moreover, chemical
denervation of sympathetic and/or sensory nerves has been demonstrated to
modulate the number of bone-resorbing osteoclasts
(6,
14). These observations
suggest that sympathetic and/or sensory innervation is required for regulating
bone metabolism under physiological and pathological conditions. It is well
known that
-adrenergic agonists can stimulate bone resorption in the
intact mouse calvaria (19).
The stimulation may be mediated by the activation of osteoclastic cells and/or
the production of osteotrophic factor by osteoblastic cells, which factor is
capable of stimulating the development of osteoclasts from their hematopoietic
precursors. Recently, we observed that epinephrine increased the expression of
osteotrophic factors, such as interleukin (IL)-6, IL-11, PGE2, and
receptor activator of NF-
B ligand (RANKL; see Refs.
16 and
29), as well as the formation
of osteoclast-like cells from mouse bone marrow cells by activating
-adrenoceptors (29).
These in vitro evidences may suggest that osteoblastic-mediated
osteoclastogenesis is regulated by sympathetic activity in
vivo.
Administration of lipopolysaccharide (LPS) has been shown to increase the
norepinephrine (NE) turnover rate in various brain areas and peripheral
tissues (1,
12). Recent experiments showed
that the intracerebroventricular injection of LPS induced a marked increase in
the level of IL-6 in the bloodstream and in IL-6 mRNA expression in the brain
and peripheral organs (20,
23). The increases in plasma
IL-6 levels by centrally injected LPS were reported to be inhibited by the
intraperitoneal administration of adrenergic antagonists, suggesting the
involvement of the NE system in the central LPS-induced IL-6 response
(11). Immobilization stress
also induced an increase in plasma IL-6 levels
(27). As in the case of the
central LPS-induced IL-6 response, depletion of NE was reported to inhibit the
stress-induced increase in plasma IL-6
(27). Thus the peripheral
sympathetic nervous system may be involved in the increase of IL-6 in
peripheral tissues induced by both centrally injected LPS and immobilization
stress.
In the present study, to determine whether sympathetic activity is involved
in bone metabolism in vivo, we examined the expression of IL-6 mRNA in
calvariae dissected from mice treated intracerebroventricularly with LPS or
subjected to restraint stress to stimulate sympathetic activity, and we
pharmacologically characterized the IL-6 expression induced by the central LPS
or physiological stress. All experiments were performed in accordance with the
guidelines for animal experiments at the School of Dentistry, Aichi-Gakuin
University.
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MATERIALS AND METHODS
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Animals and reagents. Conventional male and pregnant female ICR
mice were obtained from SLC (Hamamatsu, Shizuoka, Japan). Mice were caged in
plastic tubs covered with stainless-steel tops and containing hardwood-chip
bedding under automatically controlled conditions of temperature
(23-25°C), humidity (50 ± 10%), and a 12:12-h light-dark cycle and
were given ad libitum tap water and rodent chow. LPS from Escherichia
coli serotype 026:B6 (phenol extracted), propranolol, 6-hydroxydopamine
(6-OHDA), phentolamine, NE, isoprenaline (ISP), and phenylephrine were
purchased from Sigma (St. Louis, MO). 6-OHDA was dissolved in saline
containing 0.1% ascorbic acid. Other reagents were dissolved in saline.
Restraint stress. Five-week-old ICR mice were restrained
individually by keeping them in a 50-ml disposable syringe (with volume set
for 25-30 ml) with some holes for the desired period. Mice kept unrestrained
at room temperature served as controls. After the restraint stress, the
calvaria was removed immediately, and the total RNA was extracted by the
guanudinium-thiocyanate method
(7). In the experiment of
chemical denervation, the mice were pretreated with 6-OHDA (100 mg/kg ip) or
vehicle for 3 days before being subjected to the restraint stress. In the
experiment of receptor blockage, the mice were pretreated with propranolol (15
mg/kg sc), phentolamine (5 mg/kg ip), or saline for 10 min before the
restraint stress.
Intracerebroventricular injection of LPS. The
intracerebroventricular administration of LPS was performed by following the
method of Haley and McCormick
(13). Simply stated, ICR mice
were injected under ether anesthesia 1 mm lateral and 1 mm anteroposterior to
the bregma with a Hamilton syringe (10 µl) fitted with a 27-gauge needle.
The intracerebroventricular injection volume was 5 µl, and the injection
sites were verified by injection of the same volume of methylene blue in the
sites. After the injection of LPS, the calvariae were obtained at various
times for analysis of the expression of IL-6 mRNA. Mice were pretreated with
6-OHDA (100 mg/kg ip) or vehicle for 3 days before the injection of LPS. Other
mice were pretreated with propranolol (25 mg/kg ip), phentolamine (5 mg/kg
ip), or saline for 10 min before the injection of LPS (icv).
Analysis of mRNA levels by RT-PCR. RNA was extracted from mouse
calvaria by the guanidinium-thiocyanate method. The total RNA was solubilized
in 1 ml guanidinium thiocyanate buffer/calvaria from a mouse and then
extracted with phenol and treated with DNase I (Boehringer Mannheim). cDNA was
synthesized by using random primer and Moloney murine leukemia virus RT
(GIBCO-BRL, Grand Island, NY), and subsequent PCR amplification was done by
using synthetic gene primers specific for mouse IL-6 and mouse
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) produced from their
respective reported cDNA sequences
(2,
12). The oligonucleotide
primers were synthesized on a DNA synthesizer (Expedite model 8909; PerSeptiv
Biosystem, Cambridge, MA) and purified on a polypropylene filter (Oligo Prep
kit; Pharmacia Biotech, Uppsala, Sweden). GAPDH primers (forward primer
5'-ACCACAGTCCATGCCATCAC-3', reverse primer
5'-TCCACCACCCTTTGCTGTA-3') were used to amplify a 452-bp cDNA
fragment, and mouse IL-6 primers (forward primer
5'-GAAATGAGAAAAGAGTTGTGC-3', reverse primer
5'-ATTGGAAATTGGGGTAGGAAG-3') were used to amplify a 324-bp cDNA
fragment. PCR amplification was performed by using a GeneAmp PCR System
(Perkin-Elmer/Cetus, Norwalk, CT) under the following conditions: denaturation
at 95°C for 15 s, annealing at 55°C or 30 s, and elongation at
72°C for 30 s for the appropriate number of cycles. PCR products were
electrophoresed on a 2% NuSive GTG agarose gel (FMC BioProducts, Rockland,
ME), stained with ethidium bromide, and detected on a fluoroimage analyzer
(FluorImager 575; Molecular Dynamics, Sunnyvale, CA). All PCR data were
obtained from the measurements, which were performed in the linear range of
PCR amplification.
Analysis of IL-6 production in calvaria culture system. Calvariae
(frontal and parietal) were aseptically removed from 2- or 3-day-old mice.
They were preincubated in medium containing 100 U/ml penicillin and 100
µg/ml streptomycin for 18 h at 37°C in air with 5% CO2. Then
calvariae were treated with NE (100 µM), ISP (100 µM), or phenylephrine
(100 µM) for 24 h, and condition medium was used for analysis of IL-6
synthesis. IL-6 in condition medium was quantified using an ELISA kit (R&D
Systems, Minneapolis, MN).
Statistical analysis. All data were presented as means ±
SE. Statistical analysis was carried out by one-way or two-way ANOVA. Fisher's
protected least significant difference post hoc test was used when multiple
groups were compared (Figs. 2,
3,
4), and Student's
t-test was used when groups were compared with a single control group
(Figs. 1 and
5).

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Fig. 2. Effect of 6-hydroxydopamine (6-OHDA) on restraint stress-induced
(A) and LPS-induced (B) increases in the IL-6 mRNA levels in
mouse calvaria. a: RT-PCR analysis of mRNA obtained from mouse
calvaria. Mice were injected with either vehicle (-) or 100 mg/kg 6-OHDA (+)
for 3 days. Later (3 days), the effects of restraint stress (A) for 0
(-) or 30 (+) min and central LPS administration (B) at 0 (-) or 50
(+) ng/mouse for 60 min were examined. Arrowheads indicate the predicted sizes
of PCR products, and nos. in parentheses indicate cycles for PCR
amplification. DNA size markers ( X174-Hae III digest) are shown
on left (S). Data shown are representative of 6 similar experiments.
b: Relative expression of these increases. Values are means ±
SE of 6 mice. *P < 0.05 vs. control mice (--).
#P < 0.05 vs. control mice with 6-OHDA (-+).
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Fig. 3. Effect of propranolol on restraint stress-induced (A) and
LPS-induced (B) increases in the IL-6 mRNA levels in mouse calvaria.
a: RT-PCR analysis for mRNA obtained from mouse calvaria. Mice were
injected with either vehicle (-) or 15 mg/kg propranolol (Pro; +). Later (10
min), the effects of restraint stress (A) for 0 (-) or 30 (+) min or
central LPS administration (B) at 0 (-) or 50 (+) ng/mouse for 60 min
were examined. Arrowheads indicate the PCR production of predicted sizes, and
nos. in parentheses indicate cycles for PCR amplification. DNA size markers
( X174-Hae III digest) are shown on left (S). Data
shown are representative of 6 similar experiments. b, Relative
expression of these increases. Values are means ± SE of 6 mice.
*P < 0.01 vs. control mice (--).
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Fig. 4. Effect of phentolamine on restraint stress-induced (A) and
LPS-induced (B) increases in the IL-6 mRNA levels in mouse calvaria.
Relative expression of IL-6 was calculated by dividing the intensity of the
IL-6 band by that of the GAPDH band as determined with a fluorescent image
analyzer. Mice were injected with either vehicle (-) or 5 mg/kg phentolamine
(Ph; +). Later (10 min), the effects of restraint stress (A) for 0
(-) or 30 (+) min or central LPS administration (B) at 0 (-) or 50
(+) ng/mouse for 60 min were examined. Values are means ± SE of 6 mice.
*P < 0.01 vs. control mice (--). #P < 0.05
vs. control mice with Ph (-+).
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Fig. 1. RT-PCR analysis of mRNA obtained from calvariae of mice treated with
restraint stress (A) or icv lipopolysaccharide (LPS; B and
C). A: a, RT-PCR analysis of mRNA obtained from
mouse calvaria. Mice were subjected to a restraint stress for 0 (no stress),
15, 30, or 60 min. DNA size markers ( X174-Hae III digest) are
shown on left (S). Arrowheads indicate the predicted size of PCR
products. Nos. in parentheses indicate cycles for PCR amplification. Data
shown are representative of 6 similar experiments. b, relative
expression of these increases. The mRNA level of interleukin (IL)-6 was
calculated by dividing the intensity of the IL-6 band by that of the GAPDH
band as determined by fluorescent image analyzer. Values are means ± SE
of 6 mice. *P < 0.05 vs. nonstressed mice (0 min).
B: a, RT-PCR analysis of mRNA obtained from mouse calvaria.
Mice were treated with LPS (50 ng/mouse icv) for 0 (nontreatment), 30, 60, or
120 min. Data shown are representative of 6 similar experiments; b,
relative expression of these increases. Values are means ± SE of 6
mice. *P < 0.01 vs. nontreated mice (0 min).
C: a, mice were treated with 0 (saline), 0.5, 5, or 50
ng/mouse LPS (icv) for 60 min. Data shown are representative of 6 similar
experiments; b, relative expression of these increases. Values are
means ± SE of 6 mice. *P < 0.01 vs.
saline-treated mice (0 ng/mouse).
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Fig. 5. Changes of IL-6 synthesis in calvaria cultivated with norepinephrine (NE),
isoprenalin (ISP), or phenylephrine (PHN). Calvariae dissected from 2- or
3-day old mice were preincubated for 18 h, and then calvariae were treated
with norepinephrine (100 µM), isoprenaline (100 µM), or phenylephrine
(100 µM). Conditioned media were used for analysis of IL-6 synthesis by the
ELISA system. Values are means ± SE of 5 calvariae.
*P < 0.05 vs. control (CTRL).
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RESULTS
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The effects of restraint stress and intracerebroventricular LPS on IL-6
mRNA levels in mouse calvaria are shown in
Fig. 1. RT-PCR analysis
revealed a low level of IL-6 mRNA in the mouse calvaria, which was increased
by exposure of the mice to restraint stress for 30 min. The restraint stress
for 15 min was not sufficient to increase the IL-6 mRNA levels
(Fig. 1A). Restraint
stress for 30 min increased the plasma epinephrine level in mice
(Table 1).
Figure 1B shows the
effect of LPS (icv) on the IL-6 mRNA levels in mouse calvaria. Treatment of
mice with LPS (50 ng/mouse icv) for 60-120 min significantly increased the
IL-6 mRNA levels in the calvaria. The increase by treatment with LPS for 30
min was lower than that by treatment for 60 or 120 min
(Fig. 1B). The
dose-dependent effect of LPS on the IL-6 mRNA levels in the calvaria is shown
in Fig. 1C. By
treatment of mice with LPS (icv) for 60 min, the dose-dependent increase in
the IL-6 mRNA level was observed at a dose of 0.5-50 ng/mouse.
To determine whether the restraint stress-induced and LPS (icv)-induced
increases in the IL-6 mRNA in mouse calvaria required peripheral NE nerve
activity, we examined the effect of 6-OHDA (well known to destroy NE nerve
terminals) on these increases in IL-6 mRNA levels.
Table 1 shows the results of
ANOVA for the effects of LPS, stress, and 6-OHDA on IL-6 mRNA in mouse
calvaria. In the expression of IL-6 mRNA, there were significant differences
in the LPS group (P < 0.001) and stress group (P <
0.05) in mouse calvaria. However, there were no significant differences in the
interaction of stress and 6-OHDA, but there were significant differences in
the interaction of LPS and 6-OHDA (P < 0.001). As shown in
Fig. 2A, the 2.8-fold
increase in the IL-6 mRNA levels in calvaria by restraint stress for 30 min
was not influenced by the pretreatment of mice with 6-OHDA. Actually, this
pretreatment with 6-OHDA caused a slight increase over the level obtained by
the stress. On the other hand, the 2.6-fold increase in the IL-6 mRNA levels
caused by treatment with LPS (50 ng/mouse icv) for 60 min was inhibited by
pretreatment with 6-OHDA (Fig.
2B).
To find out whether the restraint stress-induced and LPS (icv)-induced
increases in IL-6 mRNA in mouse calvaria were mediated by adrenoceptors in the
calvaria, we examined the effect of propranolol, a
-adrenergic
antagonist, and phentolamine, an
-adrenergic antagonist, on these
increases in the IL-6 mRNA level. Tables
2 and
3 show the results of ANOVA for
the effects of LPS, stress, propranolol, and phentolamine on IL-6 mRNA in
mouse calvaria. There were significant differences in the interaction of
stress and propranolol (P < 0.05) but no differences in the
interaction of stress and phentolamine. Furthermore, there were significant
differences in the interaction of LPS and propranolol (P < 0.01)
but no significant differences in the interaction of LPS and phentolamine. As
shown in Fig. 3A,
pretreatment with propranolol (15 mg/kg ip) inhibited the restraint
stress-induced increase in the IL-6 mRNA level in mouse calvaria. Similarly,
pretreatment with propranolol (25 mg/kg ip) inhibited the LPS-induced increase
as well (Fig. 3B).
However, pretreatment with propranolol (15 mg/kg sc) did not inhibit the
LPS-induced increase (data not shown). In contrast, pretreatment with
phentolamine (5 mg/kg ip) did not affect either the restraint stress-induced
or the LPS-induced increase in IL-6 mRNA levels in mouse calvaria
(Fig. 4, A and
B).
As shown in Fig. 5,
treatment with 100 µM NE or 100 µM ISP for 24 h increased IL-6 synthesis
in mouse calvaria. However, treatment with 100 µM phenylephrine for 24 h
did not affect IL-6 synthesis.
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DISCUSSION
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It has been demonstrated that human osteoblastic and osteoclastic cells are
equipped with adrenergic receptors and neuropeptide receptors and that they
constitutively express diffusible axon guidance molecules that are known to
function as a chemoattractant and/or a chemorepellent for growing never fibers
(30-32).
These recent findings, in addition to immunohistochemical and pharmacological
findings, suggest that the extension of axons of sympathetic and peripheral
sensory nerves to osteoblastic and osteoclastic cells is required for the
dynamic neural regulation of local bone metabolism. Recently, adrenergic
stimulation was shown to increase the expression of osteotrophic factors, such
as IL-6, IL-11, PGE2, or RANKL, which is identical to osteoclast
differentiation factor, via
-adrenoceptors in osteoblastic cells
(16,
29). Furthermore, both Takeda
et al. (28) and Baldock et al.
(3) have recently provided
pharmacological genetic evidence that hypothalamic autonomic signals
proceeding via the
-adrenoceptor regulate bone mass. In association with
an increase of sympathetic nerve activity, application of stress
(21,
27,
33) and central administration
of LPS (8,
9,
18) were shown to increase the
IL-6 levels in rodents. These findings led us to evaluate the calvaria
expression of IL-6 mRNA under the sympathomimetic condition caused by central
LPS injection or restraint stress to assess the physiological significance of
sympathetic nerve activity on bone metabolism in vivo.
In the present study, we observed elevated IL-6 mRNA expression in the
calvaria of mice subjected to central LPS injection or restraint stress. The
significant elevation by central LPS injection was prevented by the
destruction of NE nerve terminals by use of 6-OHDA
(17), or by blockage of
-adrenoceptors with propranolol, suggesting that the elevation of IL-6
mRNA in the calvaria was mediated by the activation of postganglion
sympathetic nerve fibers innervating the calvaria and by
-adrenoceptors
in the calvaria. On the other hand, the elevation by restraint stress was
prevented by blockage of
-adrenoceptors but not by the destruction of NE
nerve terminals, suggesting that the elevation is mediated by elevated
secretion of epinephrine from the adrenals. In fact, restraint stress for 30
min caused a significant increase (P < 0.05) in plasma epinephrine
in comparison with the level in control mice
(Table 1). Although it is
likely that the effects of propranolol on this expression could be a result of
antagonism of
-adrenoceptors in another tissue, with a concomitant
decrease of an intermediate factor that is responsible for stimulating the
increase in IL-6 mRNA levels, the possibility may be contradicted by a direct
increase of IL-6 protein in calvaria treated with
-adrenoceptor
activation (Fig. 5). Although
several evidences demonstrated the existence of the
-adrenoceptor in
human and mouse osteoblasts
(26,
30), phenylephrine did not
increase IL-6 synthesis in mouse calvaria. These data suggested that
peripheral NE increased IL-6 synthesis via
-adrenoceptor activation.
This is the first report to demonstrate that physiological and pharmacological
stimulation of the sympathetic nervous system modulates bone metabolic
activity in vivo, as evaluated by expression of IL-6 mRNA in calvaria.
In the bone microenvironment, there is a dynamic balance between resorption
and formation that maintains skeletal homeostasis. Osteoclastic bone
resorption consists of multiple steps, such as the differentiation of
osteoclast precursor in mononuclear prefusion osteoclasts, the fusion of
prefusion osteoclasts to form multinucleate osteoclasts, and the activation of
these osteoclasts to resorb bone
(22,
25). These steps seem to
progress at the site of bone resorption under the control osteotrophic
hormones locally produced in the micro-environment
(24). Potential paracrine
mediators of osteoclast activity include monocyte-macrophage
colony-stimulating factor, tumor necrosis factor (TNF)-
, IL-1, IL-6,
IL-11, PGE2, and RANKL/osteoprotegerin ligand/TNF-related
activation-induced cytokine, all of which are capable of increasing
osteoclastogenesis. It is well known that activation of
-adrenoceptors
on osteoblastic cells can stimulate bone resorption in intact mouse calvaria
(19) and induce the expression
of osteotrophic factors, such as IL-6, IL-11, PGE2, or RANKL
(16,
29). In the present study, we
showed the in vivo sympathomimetic action on the expression of IL-6 mRNA.
These evidences suggest that the activity of sympathetic nerves has a
significant effect on osteoclastogenesis by modulation of the expression of
osteotrophic factors in osteoblastic cells and support the observation that
bone resorption in rats was reduced after sympathectomy induced by
guanethidine, which specifically destroys sympathetic adrenergic fibers
(6). Further studies should
clarify the involvement of sympathetic innervation of osteoblastic cells and
give insight into the mechanism of sympathetic regulation of bone
metabolism.
In conclusion, the findings indicate that restraint stress increased IL-6
mRNA expression via peripheral epinephrine from the adrenal glands and that
LPS (icv) increased IL-6 mRNA expression via peripheral NE from sympathetic
postganglionic fibers in the mouse calvaria. In consideration of the
physiological significance of IL-6 in bone metabolism, we propose that
sympathomimetic action on calvaria may be part of the mechanism governing bone
resorption.
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DISCLOSURES
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This study was partly supported by a grant-in-aid for Scientific Frontier
Promoted Research and by a grant-in-aid for Scientific Research from the
Ministry of Education, Science, Sport and Culture of Japan (nos. 11671861 and
14571782 to A. Togari).
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FOOTNOTES
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Address for reprint requests and other correspondence: A. Togari, Dept. of
Pharmacology, School of Dentistry, Aichi-Gakuin Univ., Nagoya 464-8650, Japan
(E-mail:
togariaf{at}dpc.aichi-gakuin.ac.jp).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
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