Shunji Kumabe

Blockade of peripheral 5HT3 receptor attenuates the formalin-induced nocifensive behavior in persistent temporomandibular joint inflammation of rat

The role of peripheral 5HT3 receptors in the orofacial nocifensive behavior induced by the injection of formalin into masseter muscle was evaluated. The behavioral activities evoked by the formalin injection exhibited a biphasic response in the rats with or without temporomandibular joint (TMJ) inflammation (CFA group or non-CFA group). The orofacial nocifensive behavioral activity was enhanced after TMJ inflammation. Systemic administration of tropisetron, 5HT3 receptor antagonist, reduced the nocifensive behavioral activities in the late phase of orofacial formalin test in CFA group, but not in non-CFA group. Local administration of tropisetron into the masseter muscle in CFA group, but not in non-CFA group also attenuated the behavioral activities in the late phase. Unexpectedly, low dose of local tropisetron reduced the nocifensive behavioral activities in the early phase of orofacial formalin test in CFA group. These data suggest that induction of TMJ inflammation causes the elevation of the orofacial nocifensive behavioral activities evoked by formalin injection into masseter muscle, and that peripheral 5HT3 receptors may play a critical role in nociception and the transmission of orofacial pain.

Effects of peripheral inflammation on activation of ERK in the rostral ventromedial medulla.

In the present study, the activation of extracellular signal-regulated kinase (ERK) in the rostral ventromedial medulla (RVM) following the injection of complete Freunds adjuvant (CFA) into the rat hindpaw was examined in order to clarify the mechanisms underlying the dynamic changes in the descending pain modulatory system after peripheral inflammation. Phospho-extracellular signal-regulated kinase-immunoreactive (p-ERK-IR) neurons were observed in the nucleus raphe magnus (NRM) and nucleus reticularis gigantocellularis pars alpha (GiA). Inflammation induced the activation of ERK in the RVM, with a peak at 7 h after the injection of CFA into the hindpaw and a duration of 24 h. In the RVM, the number of p-ERK-IR neurons per section in rats killed at 7 h after CFA injection (14.2 +/- 1.7) was significantly higher than that in the control group (4.5 +/- 0.9) [P < 0.01]. At 7 h after CFA injection, about 60% of p-ERK-IR neurons in the RVM were serotonergic neurons. The percentage of RVM serotonergic neurons that are also p-ERK positive in the rats with inflammation (20.5% +/- 2.3%) was seven times higher than that in control rats (2.7% +/- 1.4%) [P < 0.01]. These findings suggest that inflammation-induced activation of ERK in the RVM may be involved in the plasticity in the descending pain modulatory system following inflammation.

Increase of galanin-like immunoreactivity in rat hypothalamic arcuate neurons after peripheral nerve injury

Galanin and galanin receptors are widely distributed within the central nervous system, and may play important roles in pain signaling and modulation. In the present study, we examined the galanin immunoreactivity (IR) in the hypothalamus and the amygdala following peripheral nerve injury. Four weeks after the operation, the ipsilateral mechanical threshold in the spared nerve injury (SNI) group (0.87 +/- 0.33 g) was significantly lower than that in the sham group (12.53 +/- 3.41 g; P < 0.05). In the SNI group, the number of galanin-IR neurons per section in the arcuate nucleus (Arc) of the hypothalamus was 10.2 +/- 1.7, significantly higher than that in the sham group (5.6 +/- 1.0; P < 0.05). These data suggest that the galanin-ergic neurons in the Arc may be involved in the functional modulation of descending pain modulation system following peripheral nerve injury.

Taste preference changes throughout different life stages in male rats

Taste preference, a key component of food choice, changes with aging. However, it remains unclear how this occurs. To determine differences in taste preference between rats in different life stages, we examined the consumption of taste solutions and water using a two-bottle test. Male Sprague-Dawley rats of different ages were used: juvenile (3–6 weeks), young adult (8–11 weeks), adult (17–20 weeks), middle-aged (34–37 weeks), and old-aged (69–72 weeks). The intakes of the high and low concentration solutions presented simultaneously were measured. We observed that the old-aged group had lower preference ratios for 0.3 M sucrose and 0.1 M MSG in comparison with other groups. The preference ratio for 0.03 mM QHCl was higher in the middle-aged group than in the three younger groups and higher in the old-aged group than the juvenile group. The taste preferences for HCl and NaCl did not significantly differ among the age groups. The old-aged group tended to prefer high concentrations of sucrose, QHCl, NaCl, and MSG to low concentrations, indicating age-related decline in taste sensitivity. We also aimed to investigate differences between life stages in the electrophysiological responses of the chorda tympani nerve, one of the peripheral gustatory nerves, to taste stimuli. The electrophysiological recordings showed that aging did not alter the function of the chorda tympani nerve. This study showed that aging induced alterations in taste preference. It is likely that these alterations are a result of functional changes in other peripheral taste nerves, the gastrointestinal system, or the central nervous system.

Expression of Plectin-1 and Trichohyalin in Human Tongue Cancer Cells

In basal squamous cells, plectin-1 interacts with intermediate filaments, whereas trichohyalin, which is distributed primarily in the medulla and inner root sheath cells of human hair follicles, plays a role in strengthening cells during keratinization. Although both cytoskeletal proteins occur in trace amounts in human tongue epithelial cells, there are minimal data on their expression in human tongue primary cancer cells. We therefore investigated the expression of plectin-1 and trichohyalin in human tongue epithelial cell line (DOK) and tongue cancer cell line (BICR31) using western blotting and FITC-labeled immunocytochemistry techniques. DOK and BICR31 cells were cultivated to subconfluence in Dulbecco’s Modified Eagle’s Medium containing 0.4 μg/ml of hydrocortisone and 10% fetal bovine serum, and the levels of trichohyalin and plectin-1 were determined by western blot analysis and immunocytochemical staining. Trichohyalin expression was clearly observed, with no differences between DOK and BICR31 cells. Although DOK cells expressed trace levels of plectin-1, obvious plectin-1 bands were detected in western blot analyses of BICR31 cells. Immunocytochemical staining revealed that trichohyalin and plectin-1 localize in the cytoplasm. Trichohyalin was diffusely distributed in both cell lines, and colocalization of trichohyalin and cytokeratin 1/10 was observed in almost all BICR31 cells. There were no correlations between western blot and immunocytochemical data for trichohyalin. Conversely, correlations in immunochemical reactions for plectin-1 were observed. Most DOK cells showed no localization of plectin-1, but strong reactions were detected in the cytoplasm of BICR31 cells. These results indicate that trichohyalin is expressed by cancerous tongue epithelial cells during various stages of malignancy and that plectin-1 provides an index of malignancy.

Histological Alterations from Condyle Repositioning with Functional Appliances in Rats

OBJECTIVE This study was designed to assess the morphological and histological alterations of the condyle of rats undergoing forward mandibular repositioning via functional appliance. MATERIALS AND METHODS Functional appliances were mounted onto the upper jaws of rats. Morphological analysis was conducted on micro-CT images of sacrificed animals. Histological changes in condyle were examined by immunohistochemistry using proliferating cell nuclear antigen (PCNA), matrix metalloproteases (MMPs), vascular endothelial growth factor (VEGF), tissue inhibitors of matrix metalloproteinases (TIMP-1), interleukin 1b (IL-1β), Aggrecan and Type II collagen. Osteoclast activity was identified by tartrate-resistant acid phosphatase (TRAP) staining. RESULTS Morphological analysis confirmed the forward positioning of the condyles of rats by the appliance, but the position gradually returned to normal on days 14 after treatment. An increase in PCNA positive cells was observed in the posterior region of the condyles on days 7, whereas PCNA positive cells decreased in the anterior region. Aggrecan and Type II collagen localization increased in the posterior region throughout the entire period, but decreased in the anterior region on days 14. In both regions, IL-1β and VEGF localization was significantly increased for 14 days while MMPs localization was evident throughout the entire period. The TRAP positive cells were significantly elevated on days 3 and 7. CONCLUSIONS These results suggest that the functional appliance therapy induces significant morphological and histological changes in the anterior and posterior regions of the condyle and subsequently causes adaptive cellular functions such as chondrocyte differentiation and cartilage matrix formation.

A histological study of mineralised tissue formation around implants with 3D culture of HMS0014 cells in Cellmatrix Type I-A collagen gel scaffold in vitro

We cultured HMS0014 Yub621b cells within a 3D collagen gel scaffold (Cellmatrix Type I-A) and aimed to study the fate and contribution of human bone-derived mesenchymal stem cells (MSCs) in the guided bone regeneration(GBR)-engineered tissue which has developed around the titanium (Ti) test dental implant (IP) in vitro. The light microscopy (LM) and transmission electron microscopy (TEM) results of the peri-IP tissue indicated that collagen fibrils of the Cellmatrix Type I-A gel were accumulated and fabricated to provide a 3D meshwork for proliferation and differentiation of the HMS0014 cells in the top (cell) layer; mineralisation of the GBR tissue had commenced since day 1 and became markedly deposited between days 7 and 14 of the experiment. TEM observation revealed sedimentation of cement line at the periphery of the interwoven Cellmatrix fibres and fibrils in the ECM scaffold of the GBR tissue; matrix vesicle-mediated and appositional collagen-mediated mineralisation were identified in the peri-IP ECM scaffold. The fine structure study of the plurimorphic osteoblast(Ob)-like osteogeneic cells demonstrated numerous membranous organelles related with vesicular trafficking, secretion and endocytosis in the cytoplasm; well-developed cytoskeleton networks and intercellular junctional complexes were also observed. The specimens on fluorescence immunohistochemistry (IHC) by confocal laser-scanning microscopy (LSM) showed the expression of LC3 and Cx43 associated with autophagic-lysosomal degeneration pathway and signal conduction mediated with gap junctions (GJS) in maintaining tissue homeostasis of the Ob-like cells which grew and degenerated in the 3D scaffold. Results from this in vitro study suggest that Ob-like HMS0014 cells actively regulate turnover of the peri-IP ECM to recapitulate the development and formation of osteoid tissue-engineered material which might contribute to augment osseointegration around the dental implant.

Transient Receptor Potential Vanilloid Subtypei1 (TRPV1) Expression in Bone Marrow-derived Stromal Cells of Rats in vitro

We studied rat femur bone marrow stroma cells (BMSCs) and microenvironment turnover in capsaicin (CAP)-conditioned media in vitro. Proliferation, differentiation and TRPV1 expression at the mRNA level (RT-PCR) of conditioned BMSCs, and pH changes and calcium assay of the conditioned culture media were examined; the obtained data were statistically analyzed. Elapsed time-dependent cell proliferation of BMSCs peaking at day 10 was observed in alkaline phase and 37℃ microenvironment. Although there were no significant differences of proliferation in different conditions on designated experimental days, day 10 was the turning point of proliferation phase where intermittent administration of 25µm CAP resulted in the least amount of cell proliferation than other culture conditions on day 14. The present RT-PCR study revealed expression of TRPV1 mRNA during 3 to 4 days (either day 3 or day 7) after single or intermittent exposure under 25µM CAP. This study elucidated that by intermittent addition of 25µM CAP into the culture medium, the CAP-sensing TRPV1 of the rat BMSCs was activated to significantly mediate Ca (superscript 2+) influx leading to cell death, and the TRPV1 was desensitized, between day 7 and day 10 in vitro.

A Preliminary Study of Osseointegration in the Dental Implant Therapy in vitro-culture of-Culture of Mouse KUSA/A1 Cells on Titanium Plates with Different Surface Modifications

To sustain an extensive osseointegration with optimal bone-to-implant contact (BIC) is one of the essentials for a successful oral implant therapy. In the present in vitro study, we have preliminarily elucidated that KUSA/A1 cells actively proliferated and have differentiated into osteocalcin-producing osteoblast-like cells (Obs) since day 3 of culture. We then cultured the Obs on titanium (Ti) and Ti alloy plates (discs), which have commercially available surface texture and modifications (i.e., anodic oxidization: AO, hydroxyapatite coating: HA, precision blasting: SPI) as same as the current dental implants. The histology of proliferating Obs cultured on discs (Disc-AO, Disc-HA and Disc-SPI; for 180 minutes) was investigated to study the microenvironment mimic initial osseointegration of the dental implant therapy. The present SEM of 60-min Disc-AO, Disc-HA and Disc-SPI specimens demonstrated a loose distribution of spherical-to-ovoid (10 μm in diameter, avg.) Obs migrating towards concavities of the substrate. The proliferating Obs had the bulging cell body sending out many minute filopodia and some lamellipodia to attach with the substrate; few Obs with intercellular junctions were dispersedly found. In contrast, in the 180-min Disc-AO, Disc-HA and Disc-SPI specimens, we observed the cultured KUSA/AI cells actively proliferated and spread into flat, large polygonal (20 μm × 30 μm, approx.) Obs with several prominent lamellipodia and many dendritic filopodia to employ cell-to-disc and intercellular attachments; a few scattered small spherical-to-spindle shaped Obs were also observed interposing the large polymorphic Obs. On the other hand, the present immunohistochemistry of the attached Obs demonstrated the expression of F-actin (actin filaments of the cytoskeleton) in both 60-min and 180-min specimens, and CD51 ( α V integrin) only in 180-min. specimens. Hence, the present study revealed that the condition-induced KUSA/A1 cells actively proliferated and extensively adhered on different substrates yet showed similar histology. We confirmed that the present GBR engineered KUSA/AI cells to obtain extensive cell attachment on different microtextured Ti discs within 180 min, thereby we surmised that the Obs might be further regulated to precipitate mineralization matrix for contact and distant osseointegration.

Fourier Analysis of Maxillary Dental Arch Forms

Our previous principal component analysis conducting on reference points, lines and angles, and a vector-developed polar coordinate system has elucidated that the components of eigenvectors had positive relationships in the curvature of anterior teeth segment, between the protrusion of canines and degree of arch roundness, and in the length-to-width ratio of 62 maxillary dentitions, which were preliminarily classified with reference to the conventional Thompson’s morphological descriptions for dental arch forms. In the present study on morphological characters of the maxillary dentitions, we conducted a Fourier analysis on the previously obtained data. We observed that the amplitude of 2 nd , 3 rd and 4 th Fourier harmonics were closely correlated with the length-to-width ratio, curvature of the anterior teeth segment, and the curvilinear contour of maxillary dental arches. In addition, the relationships between previously estimated data and the constant value and the amplitude of the Fourier series were examined by analysis of correlation coefficients(p＜0.01). The results of the present study suggest that the morphology of maxillary dentitions consists of three essentials-the length-to-width ratio, the curvature of anterior teeth and the curvilinear contour of dental arches.