Kaori Gunjigake

Substance P stimulates late-stage rat osteoblastic bone formation through neurokinin-1 receptors.

Substance P (SP) is a widely distributed neuropeptide that works as a neurotransmitter and neuromodulator. Recently, SP receptors, particularly neurokinin-1 receptors (NK(1)-Rs) that have a high affinity for SP, have been observed not only in neuron and immune cells, but also in other peripheral cells, including bone cells. To identify the role of SP in bone formation, we investigated the expression of NK(1)-Rs in osteoblastic cells and the effects of SP on bone formation by rat calvarial osteoblastic cells. Rat calvarial osteoblastic cells were isolated and cultured for 3 weeks in alpha-MEM containing 10% serum, ascorbic acid, dexamethasone, and beta-glycerophosphate. We then investigated NK(1)-R expression, SP effects on osteoblastic bone formation, and osteocalcin mRNA expression in osteoblastic cells. RT-PCR and immunocytochemistry showed that NK(1)-R mRNA was expressed and NK(1)-R was present in 14-day, but not 7-day, cultured calvarial osteoblasts. Bone formation by cultured osteoblastic cells significantly increased after the addition of 10(-8)-10(-6)MSP. During 3 weeks of culture, the addition of SP in the first week did not significantly increase bone formation, whereas adding SP during the first and second week or all 3 weeks significantly increased calvarial osteoblastic bone formation. Furthermore, semi-quantitative RT-PCR indicated that SP stimulated osteocalcin mRNA expression in the osteoblasts at day 14 or day 21, whereas SP did not stimulated the runX2 or type I collagen mRNA expression at day 7 but stimulated them at day 14. These results indicate that SP stimulates bone formation by osteoblastic cells via NK(1)-Rs at late-stage bone formation. These effects were dependent on the expression of NK(1)-R in osteoblastic cells. Our findings suggest that SP secreted from sensory neurons may modulate bone formation after the expression of SP receptors.

Activation of satellite glial cells in rat trigeminal ganglion after upper molar extraction.

The neurons in the trigeminal ganglion (TG) are surrounded by satellite glial cells (SGCs), which passively support the function of the neurons, but little is known about the interactions between SGCs and TG neurons after peripheral nerve injury. To examine the effect of nerve injury on SGCs, we investigated the activation of SGCs after neuronal damage due to the extraction of the upper molars in rats. Three, 7, and 10 days after extraction, animals were fixed and the TG was removed. Cryosections of the ganglia were immunostained with antibodies against glial fibrillary acidic protein (GFAP), a marker of activated SGCs, and ATF3, a marker of damaged neurons. After tooth extraction, the number of ATF3-immunoreactive (IR) neurons enclosed by GFAP-IR SGCs had increased in a time-dependent manner in the maxillary nerve region of the TG. Although ATF3-IR neurons were not detected in the mandibular nerve region, the number of GFAP-IR SGCs increased in both the maxillary and mandibular nerve regions. Our results suggest that peripheral nerve injury affects the activation of TG neurons and the SGCs around the injured neurons. Moreover, our data suggest the existence of a neuronal interaction between maxillary and mandibular neurons via SGC activation.

Clinical estimation of mouth breathing

INTRODUCTION Breathing mode was objectively determined by monitoring airflow through the mouth, measuring nasal resistance and lip-seal function, and collecting information via questionnaire on the patients etiology and symptoms of mouth breathing. METHODS The expiratory airflow through the mouth was detected with a carbon dioxide sensor for 30 minutes at rest. Fifteen men and 19 women volunteers (mean age, 22.4 +/- 2.5 years) were classified as nasal breathers, complete mouth breathers, or partial mouth breathers based on the mean duration of mouth breathing. Nasal resistance, lip-sealing function, and the subjective symptoms of mouth breathing ascertained by questionnaire were statistically compared by using 1-way and 2-way analysis of variance (ANOVA) and the chi-square test in the breathing groups. RESULTS Nasal resistance was significantly (P <0.05) greater for the mouth breathers than for the nasal breathers, and significantly (P <0.05) greater for the partial mouth breathers than for the complete mouth breathers. There were no significant differences in the subjective responses to questions about mouth breathing among the 3 groups. CONCLUSIONS Detecting airflow by carbon dioxide sensor can discriminate breathing mode. Degree of nasal resistance and subjective symptoms of mouth breathing do not accurately predict breathing mode.

Correlation between the Appearance of Neuropeptides in the Rat Trigeminal Ganglion and Reinnervation of the Healing Root Socket after Tooth Extraction

The neuropeptide substance P (SP) modulates bone metabolism. This study examined the temporal appearance of the neuropeptides SP and brain-derived nerve growth factor (BDNF) and their receptors (neurokinin-1 receptor (NK1-R) and Trk B, respectively) in the rat trigeminal ganglion to investigate the role of neuropeptides in healing after tooth extraction. Rats were anesthetized and their upper right first molars were extracted; the rats were sacrificed 3 hours and 1–21 days after extraction. Their trigeminal ganglion and maxilla were removed, and cryosections were prepared and immunostained using specific antibodies against SP, BDNF, NK1-R, and Trk B. In the tooth sockets after extraction, new bone and a few SP-immunoreactive nerve fibers were first seen at day 7, and bone completely filled the sockets at day 21. In the trigeminal ganglion, the proportions of NK1-R-, BDNF-, and Trk B-immunoreactive neurons changed similarly, i.e., they initially decreased, increased rapidly to maximum levels by day 3, and then decreased gradually to control levels until 21 days. These findings suggest that the appearance of neuropeptides in the trigeminal ganglion, the reinnervation of SP-immunoreactive nerve fibers, and bone repair in the tooth socket during healing after extraction were correlated.

Hemokinin-1 competitively inhibits substance P-induced stimulation of osteoclast formation and function

Hemokinin-1 (HK-1) is a novel member of the tachykinin family that is encoded by preprotachykinin 4 (TAC4) and shares the neurokinin-1 receptor (NK1-R) with substance P (SP). Although HK-1 is thought to be an endogenous peripheral SP-like endocrine or paracrine molecule in locations where SP is not expressed, neither the distribution of HK-1 in the maxillofacial area nor the role HK-1 in bone tissue have been examined. In this study, we investigated the distribution of HK-1 in trigeminal ganglion (TG) and maxillary bone, and assessed the expression of HK-1 during osteoclast differentiation. In vivo, rat molars were loaded for 5 days using the Waldo method. In vitro, rat osteoclast-like cells were induced from bone marrow cells. HK-1 distribution and expression were examined by immunofluorescence staining and reverse transcription polymerase chain reaction (RT-PCR). In vivo, HK-1 was localized in rat TG neurons; however, the number of HK-1-positive neurons was less than that of SP-positive neurons. In the maxillary bone, nerve fibers, blood vessels, and osteocytes were immunopositive for HK-1. Furthermore, HK-1-positive immunoreactivity was found in osteoclasts on the pressure side. In vitro, PCR showed that TAC4 and NK1-R mRNA was expressed in osteoclasts as well as in bone marrow cells. Although SP (10⁻⁷ M) treatment led to an increased number of osteoclasts, HK-1 (10⁻⁷ M) treatment did not. The numbers of biotin-labeled HK-1 peptides bound osteoclasts significantly decreased upon incubation with unlabeled SP and biotin-labeled HK-1 compared with biotin-labeled HK-1 alone. These results suggest that HK-1 may not stimulate the differentiation and function of osteoclasts. SP-stimulated osteoclast formation is competitively regulated by peripheral HK-1 through NK1-Rs.

Neuropeptides modulate RANKL and OPG expression in human periodontal ligament cells

Abstract Neuropeptides, such as substance P (SP) and calcitonin gene-related peptide (CGRP), may be associated with bone remodeling in response to mechanical stress during orthodontic tooth movement. To investigate this hypothesis, we examined the effects of neuropeptides on the expression of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) in human periodontal ligament (PDL) cells under compression in vitro. PDL cells were subjected to compressive force (2.0 g/cm2) continuously in the presence or absence of SP or CGRP for 2–4 days. The expression of the SP receptor, neurokinin 1-receptor (NK1-R), in PDL cells was confirmed by RT–PCR and immunofluorescent staining. The effects of neuropeptides (SP and CGRP) on the expression of RANKL and OPG mRNA were determined using RT–PCR. PDL cells constitutively expressed NK1-R on both the mRNA and protein levels. Compressive force decreased OPG mRNA expression and increased RANKL mRNA expression. In the presence of neuropeptides, the OPG level decreased synergistically with compression. Neuropeptides stimulated RANKL expression without compression, whereas they decreased RANKL mRNA expression with compression. These results indicate that PDL cell compression induces the up-regulation of RANKL and down-regulation of OPG, whereas neuropeptides suppress the RANKL expression induced by compression. Therefore, the neuropeptides SP and CGRP may modulate bone remodeling by PDL cells during orthodontic tooth movement.

Asporin in compressed periodontal ligament cells inhibits bone formation

OBJECTIVE During orthodontic tooth movement, bone resorption and inhibition of bone formation occur on the compressed side, thereby preventing ankylosis. Periodontal ligament (PDL) cells control bone metabolism and inhibition of bone formation on the compressed side by secreting bone-formation inhibitory factors such as asporin (ASPN) or sclerostin (encoded by SOST). The aim of this study was to identify the inhibitory factors of bone formation in PDL cells. DESIGN In vitro, the changes in expression of ASPN and SOST and subsequent protein release in human PDL (hPDL) cells were assessed by semi-quantitative polymerase chain reaction (PCR), real-time PCR, and immunofluorescence in hPDL cells subjected to centrifugal force using a centrifuge (45, 90, 135, and 160 × g). In vivo, we applied a compressive force using the Waldo method in rats, and examined the distribution of ASPN or sclerostin by immunohistochemistry. RESULTS In vitro, hPDL cells subjected to 90 × g for 24h demonstrated upregulated ASPN and downregulated SOST expressions, which were confirmed by immunofluorescent staining. In addition, the formation of mineralized tissue by human osteoblasts was significantly inhibited by the addition of medium from hPDL cells cultured during compressive force as well as the addition of equivalent amounts of ASPN peptide. In vivo, asporin-positive immunoreactive PDL cells and osteoclasts were found on the compressed side, whereas few sclerostin-positive PDL cells were observed. CONCLUSIONS PDL cells subjected to an optimal compressive force induce the expression and release of ASPN, which inhibits bone formation during orthodontic tooth movement on the compressed side.

The effect of mouth breathing on chewing efficiency

OBJECTIVE To examine the effect of mouth breathing on chewing efficiency by evaluating masticatory variables. MATERIALS AND METHODS Ten adult nasal breathers with normal occlusion and no temporomandibular dysfunction were selected. Subjects were instructed to bite the chewing gum on the habitual side. While breathing through the mouth and nose, the glucide elution from the chewing gum, number of chewing strokes, duration of chewing, and electromyography (EMG) activity of the masseter muscle were evaluated as variables of masticatory efficiency. RESULTS The durations required for the chewing of 30, 60, 90, 120, 180, and 250 strokes were significantly (P < .05) longer while breathing through the mouth. There was no significant difference in the glucide elution rate (%) for each chewing stroke between nose and mouth breathings. The glucide elution rates for 1- and 3-minute chewing were significantly (P < .05) lower while breathing through the mouth. However, there was no significant difference in the glucide elution rate for 5-minute chewing between nose and mouth breathings. While chewing for 1, 3, and 5 minutes, the chewing stroke and EMG activity of the masseter muscle were significantly (P < .05) lower during mouth breathing. CONCLUSIONS It takes a longer amount of time to complete chewing to obtain higher masticatory efficiency when breathing through the mouth. Therefore, mouth breathing will decrease the masticatory efficiency if the duration of chewing is restricted in everyday life.

Nerve Growth Factor Involves Mutual Interaction between Neurons and Satellite Glial Cells in the Rat Trigeminal Ganglion

Nerve growth factor (NGF) plays a critical role in the trigeminal ganglion (TG) following peripheral nerve damage in the oral region. Although neurons in the TG are surrounded by satellite glial cells (SGCs) that passively support neural function, little is known regarding NGF expression and its interactions with TG neurons and SGCs. This study was performed to examine the expression of NGF in TG neurons and SGCs with nerve damage by experimental tooth movement. An elastic band was inserted between the first and second upper molars of rats. The TG was removed at 0–7 days after tooth movement. Using in situ hybridization, NGF mRNA was expressed in both neurons and SGCs. Immunostaining for NGF demonstrated that during tooth movement the number of NGF-immunoreactive SGCs increased significantly as compared with baseline and reached maximum levels at day 3. Furthermore, the administration of the gap junction inhibitor carbenoxolone at the TG during tooth movement significantly decreased the number of NGF-immunoreactive SGCs. These results suggested that peripheral nerve damage may induce signal transduction from neurons to SGCs via gap junctions, inducing NGF expression in SGCs around neurons, and released NGF may be involved in the restoration of damaged neurons.

Neurokinin B activates the formation and bone resorption activity of rat osteoclasts.

Neurokinin B (NKB) is a neuropeptide in the tachykinin family that acts as a neurotransmitter and neuromodulator, primarily in the central nervous system. The distribution and role of NKB and its receptor, the neurokinin-3 receptor (NK-3R), in peripheral tissues are poorly understood. In this study, we investigated the distribution of NKB and NK-3R in peripheral tissues as well as the role of NKB in bone metabolism, especially in osteoclast formation and bone resorption activity through NK-3R. The distributions of NKB in intact rat neurons of the trigeminal ganglion (TG) and in axons of periodontal tissue were investigated by immunohistochemistry. Osteoclasts from cultured rat bone marrow cells were used to examine the distribution of NK-3R by immunocytochemistry and RT-PCR and to investigate the effects of NKB on the resorption activity of osteoclasts on ivory slices. We found that NKB immunopositive neurons were localized in the rat TG and that NKB immunopositive axons were distributed in periodontal tissues. Immunoreactivity for NK-3R was found in cultured osteoclasts, and NK-3R mRNA expression in the osteoclasts was confirmed by RT-PCR. The addition of NKB significantly increased the number of osteoclasts and the resorption area compared with the control. These findings suggest that NKB was localized in peripheral neurons and may involve the activation of osteoclast formation and bone resorption through NK-3R.