Cisplatin induces TRPA1-mediated mechanical allodynia in the oral mucosa.

OBJECTIVE: Cisplatin, a platinum-based anticancer drug, produces reactive oxygen species (ROS) in many cell types and induces mechanical allodynia in the hands and/or feet (chemotherapy-induced painful neuropathy: CIPN). In this study, we examined the possibility of inducing neuropathy in the oral region using oral keratinocytes and rats. METHODS: Human oral keratinocytes (HOKs) were used to evaluate ROS generation after cisplatin application by a ROS-reactive fluorescent assay. In rats, after cisplatin administrations (two times), the trigeminal ganglion (TG) was investigated by electron microscopy and quantitative RT-PCR. Using our proprietary assay system, oral pain-related behaviors were observed in cisplatin-treated rats. RESULTS: In rats, cisplatin administration reduced food intake and body weight. In electron microscopic analysis, glycogen granules in the TG were depleted following administration, although organelles were intact. In HOK cells, cisplatin significantly increased ROS generation with cell death, similar to glycolysis inhibitors. Cisplatin administration did not show any effects on Trpa1 mRNA levels in the TG. However, the same procedure induced hypersensitivity to mechanical stimulation and the TRPA1 agonist allyl isothiocyanate in the oral mucosa. Mechanical hypersensitivity was inhibited by the antioxidative drug α-lipoic acid and the TRPA1 antagonist HC-030031, similar to that of the hind paw. CONCLUSION: The present findings suggest that cisplatin induces TRPA1-mediated CIPN due to ROS generation in the oral region. This study will provide a better understanding of persistent oral pain in cancer patients.

Vesicular nucleotide transporter mediates adenosine triphosphate release in compressed human periodontal ligament fibroblast cells and participates in tooth movement-induced nociception in rats.

OBJECTIVE: Pain control is imperative in orthodontic treatment. Adenosine triphosphate (ATP) is a key mediator released from periodontal ligament cells that excites nociceptive nerve endings. Vesicular nucleotide transporter (VNUT), encoded by the Solute carrier family 17 member 9 (SLC17A9) gene, participates in ATP uptake into secretory vesicles; thus, it may mediate tooth movement-induced pain. In the present study, we examined whether VNUT in periodontal ligament cells participates in tooth movement-induced nociception. DESIGN: Expression levels of SLC17A9, connexin 43, and pannexin 1 in human periodontal ligament fibroblasts (HPDLFs) were examined by quantitative reverse transcription-polymerase chain reaction. Mechanical force via centrifugation-induced ATP release was measured using an ATP bioluminescence assay. Inhibitors were used to evaluate the role of ATP transporters. Face-grooming behaviors were assessed as indicators of nociceptive responses after experimental tooth movement in rats, as well as the effects of drugs for the pain-like behavior. RESULTS: After HPDLFs underwent mechanical stimulation by centrifugation, SLC17A9 mRNA expression in the cells was significantly upregulated. Increased ATP release from HPDLFs after mechanical stimulation was suppressed by treatment with clodronic acid, a VNUT inhibitor, at concentrations of 0.1 and 1.0 µM. In rats, face-grooming behaviors (indicators of nociception) were significantly increased on day 1 after experimental tooth movement. Increased face-grooming behaviors were suppressed by systemic administration of clodronic acid (0.1 mg/kg). CONCLUSIONS: These results indicate that release of ATP from periodontal ligament cells via VNUT is important for nociceptive transduction during orthodontic treatment. Thus, VNUT may provide a novel drug target for tooth movement-induced pain.

Mash1-expressing cells could differentiate to type III cells in adult mouse taste buds.

The gustatory cells in taste buds have been identified as paraneuronal; they possess characteristics of both neuronal and epithelial cells. Like neurons, they form synapses, store and release transmitters, and are capable of generating an action potential. Like epithelial cells, taste cells have a limited life span and are regularly replaced throughout life. However, little is known about the molecular mechanisms that regulate taste cell genesis and differentiation. In the present study, to begin to understand these mechanisms, we investigated the role of Mash1-positive cells in regulating adult taste bud cell differentiation through the loss of Mash1-positive cells using the Cre-loxP system. We found that the cells expressing type III cell markers-aromatic L-amino acid decarboxylase (AADC), carbonic anhydrase 4 (CA4), glutamate decarboxylase 67 (GAD67), neural cell adhesion molecule (NCAM), and synaptosomal-associated protein 25 (SNAP25)-were significantly reduced in the circumvallate taste buds after the administration of tamoxifen. However, gustducin and phospholipase C beta2 (PLC beta2)-markers of type II taste bud cells-were not significantly changed in the circumvallate taste buds after the administration of tamoxifen. These results suggest that Mash1-positive cells could be differentiated to type III cells, not type II cells in the taste buds.

Hey1 and Hey2 are differently expressed during mouse tooth development.

The Hey family (also known as Chf, Herp, Hesr, and Hrt) is a set of Hairy/Enhancer of Split-related basic helix-loop-helix type transcription factors. Hey1, Hey2, and HeyL have been identified in mammals. Although Hey proteins are known to regulate cardiovascular development, muscle homeostasis, osteogenesis, neurogenesis, and oncogenesis, their roles in tooth development have been largely obscure. Therefore, this study aimed to clarify detailed spatiotemporal expression patterns of Hey1 and Hey2 in developing molars and incisors of mice by section in situ hybridization. Hey1 and Hey2 were not significantly expressed in tooth germs at epithelial thickening, bud, and cap stages during molar development. In the dental epithelium in molars at the bell stage and incisors, Hey2 transcripts were restricted to the undifferentiated inner enamel epithelium and down-regulated in preameloblasts and ameloblasts. On the other hand, Hey1 was mainly expressed in preameloblasts and down-regulated in differentiated ameloblasts. Both genes were not significantly expressed in other dental epithelial tissues, including the outer enamel epithelium, stellate reticulum, and stratum intermedium cells. In the dental mesenchyme, Hey1 was intensely transcribed in the subodontoblastic layer of the dental pulp in both molars and incisors, whereas Hey2 was barely detectable in mesenchymal components. Our data implied that Hey2 function is restricted to transient amplifying cells of the ameloblast cell lineage and that Hey1 plays a role in the composition of the subodontoblastic layer, in addition to ameloblast differentiation. These findings provide novel clues for the better understanding of tooth development.

Lysophosphatidylcholine acyltransferase 4 is involved in chondrogenic differentiation of ATDC5 cells.

Glycerophospholipids have important structural and functional roles in cells and are the main components of cellular membranes. Glycerophospholipids are formed via the de novo pathway (Kennedy pathway) and are subsequently matured in the remodeling pathway (Lands' cycle). Lands' cycle consists of two steps: deacylation of phospholipids by phospholipases A2 and reacylation of lysophospholipids by lysophospholipid acyltransferases (LPLATs). LPLATs play key roles in the maturation and maintenance of the fatty acid composition of biomembranes, and cell differentiation. We examined whether LPLATs are involved in chondrogenic differentiation of ATDC5 cells, which can differentiate into chondrocytes. Lysophosphatidylcholine acyltransferase 4 (LPCAT4) mRNA expression and LPCAT enzymatic activity towards 18:1-, 18:2-, 20:4-, and 22:6-CoA increased in the late stage of chondrogenic differentiation, when mineralization occurred. LPCAT4 knockdown decreased mRNA and protein levels of chondrogenic markers as well as Alcian blue staining intensity and alkaline phosphatase activity in ATDC5 cells. These results suggest that LPCAT4 plays important roles during the transition of chondrocytes into hypertrophic chondrocytes and/or a mineralized phenotype.

ADAM17 regulates TNF-α expression upon lipopolysaccharide stimulation in oral keratinocytes.

A disintegrin and metalloprotease 17 (ADAM17) is a tumor necrosis factor (TNF)-converting enzyme and was first identified as the enzyme that cleaves the prodomain of TNF-α, a proinflammatory cytokine that plays a central role in immune regulation and a variety of inflammatory responses in destructive periodontal disease. The aim of the present study was to verify the presence of ADAM17 in the gingival epithelium and elucidate its involvement in the release of TNF-αin oral keratinocytes. Immunohistochemical analyses of ADAM17 were performed in gingival tissues obtained from patients and in human oral keratinocytes (HOKs). Additionally, levels of TNF-α and ADAM17 in HOKs exposed to lipopolysaccharide (LPS) were assessed using enzyme-linked immunosorbent assays. Moreover, the effects of ADAM17 inhibitor, matrix metalloproteinase (MMP) inhibitor, and ADAM17 siRNA on TNF-α concentration were assessed. Strong immunoreactivity for ADAM17 was observed in the epithelium of the inflamed gingival tissues and in HOKs. Furthermore, treatment with either ADAM17 inhibitor or ADAM17 siRNA inhibited the generation of TNF-α induced by LPS in HOKs. The present study demonstrates that ADAM17 is strongly expressed in the epithelium of gingival tissues and suggests that ADAM17 may be a key enzyme that regulates the generation of TNF-α in oral keratinocytes.

A spatial association between odontomas and the gubernaculum tracts.

OBJECTIVES: The purpose of this study was to identify the spatial relationship and/or association between odontomas and the gubernaculum tract or the dental sac and the characteristic findings for radiographic diagnosis of odontomas. STUDY DESIGN: The visualizations of the odontomas and the gubernaculum tract were retrospectively analyzed using cone beam computed tomography or multidetector computed tomography. RESULTS: Most of odontomas were within the gubernaculum tract or dental sac of unerupted permanent teeth on computed tomography. In some odontomas, the gubernaculum tract existed as a well-defined low density tract extending from the top of odontomas on computed tomography. CONCLUSIONS: A close spatial relationship and/or association between odontomas and the gubernaculum tract or dental sac on computed tomography may be used as one of the criteria for radiographic diagnosis of odontomas. Development of odontomas may be associated with the gubernaculum tract or dental sac of unerupted permanent teeth.

Detection and imaging characteristics of the gubernacular tract in children on cone beam and multidetector computed tomography.

PURPOSE: To elucidate the appearance and imaging characteristics of the gubernacular tract (GT) during the growth stage of children. Furthermore, this study evaluated the significance of the appearance of the GT. STUDY DESIGN: The visualizations of the GT were retrospectively analyzed by using panoramic radiographs and computed tomography (CT) in children. RESULTS: In patients with normal eruption who had unerupted permanent teeth, except maxillary central supernumerary teeth, the GT was clearly visualized as a well-defined low-density tract on CT but not on panoramic radiographs. In patients with obstructive eruption, including impaction, the GT was deformed and not visible on CT. CONCLUSIONS: This paper describes the frequency of detection and appearance of the GT in unerupted teeth. Preliminary data suggest that any alteration to the GT may be used to predict abnormal eruption of permanent teeth.

Novel methods of applying direct chemical and mechanical stimulation to the oral mucosa for traditional behavioral pain assays in conscious rats.

BACKGROUND: Stomatitis induces severe and painful hypersensitivity to pungency and physical contact during meals. Many studies have used anesthetized animals to examine evoked nociception in the oral mucosa, but no reports have used traditional behavioral assays to evaluate nociception in conscious animals. NEW METHODS: We developed two new methods of applying chemical or mechanical stimulation directly to the oral mucosa of the mandibular vestibule of conscious rats. Nociceptive evaluations were performed by measuring facial grooming time and the head withdrawal threshold to von Frey stimulations. (1) For the intraoral dropping method, rat mucosa was transiently exposed by hand, and a drop of a pungent solution was applied. (2) For the stable intraoral opening method, rat mucosa was long-term exposed following piercing surgery of the mental skin after habitual training for 2-3 weeks. RESULTS: In the intraoral dropping method, the application of 100 µM capsaicin or 100 mM allyl isothiocyanate prolonged mouth-rubbing time. Capsaicin-induced mouth-rubbing time was further enhanced following the development of an acetic acid-induced ulcer. The stable intraoral opening method enabled stable measurements of the mechanical withdrawal threshold in the oral mucosa of conscious rats. Ulcer development decreased the mechanical threshold, whereas topical lidocaine treatment increased the threshold. COMPARISON WITH EXISTING METHODS: These new methods enable the evaluations of motivational nocifensive behaviors in response to intraoral stimulations without any anesthetic effects. CONCLUSIONS: The intraoral dropping and stable intraoral opening methods can be used in combination with traditional behavioral assays to evaluate nociception in the oral mucosa of conscious rats.

Can the neurovascular compression volume of the trigeminal nerve on magnetic resonance cisternography predict the success of local anesthetic block after initial treatment by the carbamazepine?

OBJECTIVES: Whether NVC volume on magnetic resonance (MR) cisternography might be related to the success of local anesthetic block by tetracaine (TNB) as an additional treatment after carbamazepine (CBZ) treatments in patients with trigeminal neuralgia (TN) was evaluated. STUDY DESIGN: Detectable NVC volumes were measured from MR cisternography in 65 patients with TN treated by TNB after CBZ treatments. The correlation between the success of TNB and the NVC volume or the improvement in pain by CBZ was evaluated retrospectively. RESULTS: A significant difference was found between the improvement in pain by CBZ and the success of TNB, but not between NVC volume on MR cisternography and the success of TNB. CONCLUSIONS: The present results suggest that the success of CBZ as initial treatment, but not NVC volume on MR cisternography, may be a significant predictor of the success of TNB as additional therapy in patients with TN.

Identification of peripheral vessels in oral and maxillofacial regions on magnetic resonance angiography obtained using a balanced steady-state free-precession sequence with a time-spatial labeling inversion pulse and using fresh blood imaging.

OBJECTIVE: To elucidate the characteristics of visualizing thin main peripheral vessels in oral and maxillofacial regions of 3-dimensional magnetic resonance angiography (MRA) using a balanced steady-state free-precession (SSFP) sequence with a time-spatial labeling inversion pulse (time-SLIP) and using fresh blood imaging (FBI). STUDY DESIGN: The conspicuity of blood vessels and the characteristics on MRA using SSFP with a time-SLIP was compared with those on MRA using FBI in 20 healthy participants. RESULTS: The conspicuity of the main peripheral arteries was significantly higher on MRA using SSFP with a time-SLIP than on MRA using FBI. MRA scans using SSFP were obtained in all participants, and scans using FBI were obtained in 16 of 20 participants. An electrocardiogram was unnecessary when using SSFP but was necessary when using FBI. CONCLUSIONS: MRA obtained using SSFP with a time-SLIP is a useful technique to visualize thin main peripheral arteries in the oral and maxillofacial regions without contrast medium.

Occurrence of silk stitch abscess after surgery in patients with oral squamous cell carcinoma.

OBJECTIVES: To elucidate the predisposing factors and clinical characteristics related to the occurrence of stitch abscess after surgery in patients with oral squamous cell carcinoma (SCC). PATIENTS AND METHODS: The subjects were 232 patients who underwent excision and/or reconstruction and/or neck dissection for oral SCC using silk sutures for high ligation of the blood vessels. Detection rates and characteristics of patients with stitch abscess were retrospectively evaluated by comparing patients with and without stitch abscesses after surgery diagnosed by ultrasonography and findings of various modalities in 232 patients. Several echogenic dots with subtle acoustic shadows in a hypoechoic mass were identified as the characteristic findings of stitch abscess on US. The patient groups with and without stitch abscess were compared with respect to various factors to identify those that predispose to the occurrence of stitch abscess. The factors analyzed included patients' sex and age, chemotherapy treatment, radiotherapy treatment, the presence of a history of allergy, and blood test results. RESULTS: A significant correlation was found between the occurrence of stitch abscess and age, liver function abnormalities on blood tests, and the presence of a history of allergy. Multiple stitch abscesses clearly tended to occur more often than single ones in patients with stitch abscess. CONCLUSIONS: The occurrence of stitch abscesses was related to age, liver dysfunction, and/or the presence of allergies. When diagnosing stitch abscess, the occurrence of multiple stitch abscesses is important.

p130Cas, Crk-associated substrate, plays important roles in osteoclastic bone resorption.

p130Cas, Crk-associated substrate (Cas), is an adaptor/scaffold protein that plays a central role in actin cytoskeletal reorganization. We previously reported that p130Cas is not tyrosine-phosphorylated in osteoclasts derived from Src-deficient mice, which are congenitally osteopetrotic, suggesting that p130Cas serves as a downstream molecule of c-Src and is involved in osteoclastic bone resorption. However, the physiological role of p130Cas in osteoclasts has not yet been confirmed because the p130Cas-deficient mice displayed embryonic lethality. Osteoclast-specific p130Cas conditional knockout (p130Cas(ΔOCL-) ) mice exhibit a high bone mass phenotype caused by defect in multinucleation and cytoskeleton organization causing bone resorption deficiency. Bone marrow cells from p130Cas(ΔOCL-) mice were able to differentiate into osteoclasts and wild-type cells in vitro. However, osteoclasts from p130Cas(ΔOCL-) mice failed to form actin rings and resorb pits on dentine slices. Although the initial events of osteoclast attachment, such as ß3-integrin or Src phosphorylation, were intact, the Rac1 activity that organizes the actin cytoskeleton was reduced, and its distribution was disrupted in p130Cas(ΔOCL-) osteoclasts. Dedicator of cytokinesis 5 (Dock5), a Rho family guanine nucleotide exchanger, failed to associate with Src or Pyk2 in osteoclasts in the absence of p130Cas. These results strongly indicate that p130Cas plays pivotal roles in osteoclastic bone resorption.

Criteria and limitations for selecting a sagittal split ramus osteotomy for patients with skeletal mandibular prognathism and open bite.

PURPOSE: The purpose of this study is to elucidate the criteria and limitations for selecting a sagittal split ramus osteotomy (SSRO) alone for patients with skeletal mandibular prognathism and open bite. STUDY DESIGN: The relationship between the mandibular relapse after 1 year and the extent of vertical movement following SSRO alone was investigated for patients with skeletal mandibular prognathism and open bite and the criteria were determined. RESULTS: The limitation in SSRO alone was found to be 3-mm vertical movement at the gonion. There was a significant difference in the mandibular relapse between the patients with >3-mm change and <3-mm change. CONCLUSIONS: For patients with skeletal mandibular prognathism and open bite, if the vertical movement at the gonion is <3 mm, SSRO alone could be an effective treatment. However, if the movement is >3 mm, SSRO alone should be avoided and bimaxillary osteotomies may be a preferable treatment.

Magnetic resonance angiography using fresh blood imaging in oral and maxillofacial regions.

The present paper provides general dentists with an introduction to the clinical applications and significance of magnetic resonance angiography (MRA) in the oral and maxillofacial regions. Specifically, the method and characteristics of MRA are first explained using the relevant MR sequences. Next, clinical applications to the oral and maxillofacial regions, such as identification of hemangiomas and surrounding vessels by MRA, are discussed. Moreover, the clinical significance of MRA for other regions is presented to elucidate future clinical applications of MRA in the oral and maxillofacial regions.

Reflection of ¹8F-FDG accumulation in the evaluation of the extent of periapical or periodontal inflammation.

OBJECTIVES: To elucidate whether fluorine-18-labeled ((18)F) fluoro-2-deoxy-d-glucose (FDG) accumulation can reflect the extent of periodontal inflammation, periapical inflammation, or dental caries. STUDY DESIGN: (18)F-FDG accumulations on positron emission tomography (PET)-computed tomography (CT) were retrospectively compared with the size of the bone resorption areas caused by periodontal inflammation, periapical inflammation, or dental caries on panoramic radiographs, CT, and magnetic resonance imaging (MRI) in 44 subjects. RESULTS: A significant correlation was found between the size of the bone resorption area caused by periodontal (r = 0.595, P < .01) or periapical (r = 0.560, P < .01) inflammation and the highest standardized uptake value (SUVmax) of (18)F-FDG accumulation. A significant correlation was found between the periodontal (r = 0.622, P < .01) or periapical (r = 0.394, P < .01) inflammatory findings on MRI and the SUVmax of (18)F-FDG accumulation. The SUVmax of (18)F-FDG around most teeth with caries was under 1.5. CONCLUSIONS: (18)F-FDG accumulation reflects the extent of dental inflammation, not dental caries.

Magnetic resonance angiography with fresh blood imaging for identification of hemangiomas and blood vessels around hemangiomas in oral and maxillofacial regions.

OBJECTIVES: To evaluate fresh blood imaging (FBI), a magnetic resonance imaging technique that does not use contrast, for identifying hemangiomas and feeding arteries in the oral and maxillofacial regions. STUDY DESIGN: For 16 patients with hemangiomas, FBI visualizations of hemangiomas and feeding arteries in the oral and maxillofacial regions were compared with those from 3-dimensional (3D) phase-contrast magnetic resonance angiography (PC-MRA). Comparisons were based on the conspicuities of blood vessels and the 3D relationships of hemangiomas with the surrounding blood vessels. RESULTS: The conspicuity of hemangiomas, feeding arteries, and blood vessels were significantly better with FBI than with PC-MRA. After differentiating arteries from veins, 3D visualizations of hemangiomas and arteries or veins could be performed with FBI. CONCLUSIONS: FBI is a useful method in oral and maxillofacial regions for identifying the relationships between hemangiomas and the surrounding arteries or veins.

Effect of bone quality and position of the inferior alveolar nerve canal in continuous, long-term, neurosensory disturbance after sagittal split ramus osteotomy.

OBJECTIVES: To examine the relationship between the anatomical position and the bone quality of the inferior alveolar nerve (IFAN) canal and long-term neurosensory disturbance (NSD) of the IFAN after a sagittal split ramus osteotomy (SSRO). MATERIAL AND METHODS: CT images were taken of patients with mandibular prognathism. The location of the IFAN and the bone density around the IFAN were measured on CT images. Whether NSD at 6 months and 1 year after SSRO was related to the position and bone quality of the IFAN canal was analyzed. RESULTS: Significant correlations were found between the anatomical position and the bone quality of the IFAN canal and the period to the resolution of NSD of the IFAN from 3 months to 1 year after SSRO. If the HU values around the IFAN were greater than 300 HU and/or the distance from the buccal aspect of the IFAN canal to the outer buccal cortical margin was less than 6 mm, NSD of the IFAN at 1 year after SSRO was significantly increased. CONCLUSION: It is important to pay particular attention to the anatomical position of the IFAN and the bone quality around the IFAN to predict long-term NSD of the IFAN after SSRO.

Noninvasive identification of peripheral vessels of oral and maxillofacial regions by using electrocardiography-triggered three-dimensional fast asymmetric spin-echo sequences.

OBJECTIVES: The aim of this study was to evaluate the 3-dimensional images of thinner main peripheral vessels in oral and maxillofacial regions made without contrast medium by using a new technique, fresh blood imaging (FBI). A second objective was to discern arteries from veins by using the combination of FBI with the subtraction technique. STUDY DESIGN: Images from FBI were compared with those from 3-dimensional phase-contrast magnetic resonance angiography (MRA) of blood vessels in 20 healthy subjects. All images were scored for visualization and image quality of the main blood vessels. In addition, appropriate flow-spoiled gradient pulses were applied to differentiate arteries from veins in the peripheral vasculature using a combination of FBI sequences and subtraction between systole- and diastole-triggered images. RESULTS: The scores of MRA using FBI for the visualization of thin blood vessels were significantly better than those using phase contrast, whereas scores for the visualization of main blood vessels were equal. Additionally, we succeeded in our initial attempt to differentiate arteries from veins with a reasonable acquisition time. CONCLUSIONS: Our initial experience shows that FBI could be a useful method to identify 3-dimensional vasculature and to differentiate arteries from veins among thinner peripheral vessels in the oral and maxillofacial regions without using contrast medium.

Correlation of mandibular bone quality with neurosensory disturbance after sagittal split ramus osteotomy.

Our aim was to find out whether the quality of bone around the inferior alveolar nerve is correlated with neurosensory disturbance to the nerve after sagittal split ramus osteotomy (SSRO) in patients with mandibular prognathism. Computed tomograms (CT) were taken of 35 patients with mandibular prognathism and 35 without. To assess the density of bone around the inferior alveolar nerve, the width of the buccal cortical bone in the mandibular second molar regions was measured on CT. The Hounsfield units (HU) in the same regions were also measured. The number of HU in the mandible around the second molar regions was significantly higher (p<0.01) in those with neurosensory disturbance (p<0.01). The quality of bone measured by HU is associated with an increased risk of neurosensory disturbance, but the width of buccal bone is not.