Local Irradiation of Mouse Tooth Germ Gives Insight into the Direct Effects of Irradiation on Root Development.

To elucidate the mechanism underlying the failure of root formation after irradiation, we established a method of local irradiation of the molar tooth germ and demonstrated that radiation directly affected dental root development. In the current study, to locally irradiate the lower first molars of 5-day-old C57BL/6J mice, we used lead glass containing a hole as a collimator. We confirmed that our local irradiation method targeted only the tooth germ. The irradiated root was immature in terms of apical growth, and dentin formation was irregular along the outside of the root apices. Moreover, calcified tissue apically surrounded Hertwig's epithelial root sheath, which disappeared abnormally early. This method using a local irradiation experimental model will facilitate research into radiation-induced disorders of dental root formation.

Characterization of proliferation, differentiation potential, and gene expression among clonal cultures of human dental pulp cells.

Mesenchymal stem cells are a highly promising source of cells for regeneration therapy because of their multilineage differentiation potential. However, distinct markers for mesenchymal stem cells are not well-established. To identify new candidate marker genes for multipotent human dental pulp stem cells, we analyzed the characteristics and gene expression profiles of cell clones obtained from a single dental pulp specimen derived from an 11-year-old female patient. Fifty colony-forming single cell-derived clones were separately cultured until the cessation of growth. These clones varied in their proliferation abilities and surface marker (STRO-1 and CD146) expression patterns, as well as their odontogenic, adipogenic, and chondrogenic differentiation potentials. Four clones maintained their original differentiation potentials during long-term culture. Gene expression profile by DNA microarray analysis of five representative clones identified 1227 genes that were related to multipotency. Ninety of these 1227 genes overlapped with genes reportedly involved in 'stemness or differentiation'. Based on the predicted locations of expressed protein products and large changes in expression levels, 14 of the 90 genes were selected as candidate dental pulp stem cell markers, particularly in relation to their multipotency characteristics. This characterization of cell clones obtained from a single specimen of human dental pulp provided information regarding new candidate marker genes for multipotent dental pulp stem cells, which could facilitate efficient analysis or enrichment of multipotent stem cells.

Chemokine ligand 2/chemokine receptor 2 signaling in the trigeminal ganglia contributes to inflammatory hyperalgesia in rats.

This study investigated the functional significance of hyperalgesia in the CCL2/CCR2 signaling system in trigeminal ganglion (TG) neurons following inflammation. Inflammation was induced by injection of complete Freund's adjuvant (CFA) into the whisker pad of rats. The escape threshold from mechanical stimulation applied to the whisker pad 2days later was significantly lower in CFA-treated rats than in naïve rats. Fluorogold (FG) labeling was used to identify the TG neurons innervating the whisker pad. FG-labeled TG neurons were immunoreactive for CCL2/CCR2. The mean number of CCL2/CCR2-immunoreactive small/medium-diameter TG neurons was significantly higher in inflamed rats than in naïve rats. Using whole-cell patch-clamp experiments in small-diameter TG neurons, the threshold current of FG-labeled TG neurons in inflamed rats was significantly decreased compared to naïve rats. The number of spike discharges during current injections by FG-labeled TG neurons in inflamed rats was significantly increased compared to naïve rats. These characteristic effects were abolished by co-application of a CCL2 receptor antagonist. The present study provides evidence that CCL2 enhances the excitability of small-diameter TG neurons following facial skin inflammation via the upregulation of CCR2. These findings suggest that ganglionic CCL2/CCR2 signaling is a therapeutic target for the treatment of trigeminal inflammatory hyperalgesia.

From regenerative dentistry to regenerative medicine: progress, challenges, and potential applications of oral stem cells.

Adult mesenchymal stem cells (MSCs) and epithelial stem cells play essential roles in tissue repair and self-healing. Oral MSCs and epithelial stem cells can be isolated from adult human oral tissues, for example, teeth, periodontal ligament, and gingiva. Cocultivated adult oral epithelial stem cells and MSCs could represent some developmental events, such as epithelial invagination and tubular structure formation, signifying their potentials for tissue regeneration. Oral epithelial stem cells have been used in regenerative medicine over 1 decade. They are able to form a stratified cell sheet under three-dimensional culture conditions. Both experimental and clinical data indicate that the cell sheets can not only safely and effectively reconstruct the damaged cornea in humans, but also repair esophageal ulcer in animal models. Oral MSCs include dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), stem cells from apical papilla (SCAP), periodontal ligament stem cells (PDLSCs), and mesenchymal stem cells from gingiva (GMSCs). They are widely applied in both regenerative dentistry and medicine. DPSCs, SHED, and SCAP are able to form dentin-pulp complex when being transplanted into immunodeficient animals. They have been experimentally used for the regeneration of dental pulp, neuron, bone muscle and blood vessels in animal models and have shown promising results. PDLSCs and GMSCs are demonstrated to be ideal cell sources for repairing the damaged tissues of periodontal, muscle, and tendon. Despite the abovementioned applications of oral stem cells, only a few human clinical trials are now underway to use them for the treatment of certain diseases. Since clinical use is the end goal, their true regenerative power and safety need to be further examined.

Brain-derived neurotrophic factor enhances the excitability of small-diameter trigeminal ganglion neurons projecting to the trigeminal nucleus interpolaris/caudalis transition zone following masseter muscle inflammation.

BACKGROUND: The trigeminal subnuclei interpolaris/caudalis transition zones (Vi/Vc) play an important role in orofacial deep pain, however, the role of primary afferent projections to the Vi/Vc remains to be determined. This study investigated the functional significance of hyperalgesia to the brain-derived neurotrophic factor (BDNF)-tyrosine kinase B (trkB) signaling system in trigeminal ganglion (TRG) neurons projecting to the Vi/Vc transition zone following masseter muscle (MM) inflammation. RESULTS: The escape threshold from mechanical stimulation applied to skin above the inflamed MM was significantly lower than in naïve rats. Fluorogold (FG) labeling was used to identify the TRG neurons innervating the MM, while microbeads (MB) were used to label neurons projecting to the Vi/Vc region. FG/MB-labeled TRG neurons were immunoreactive (IR) for BDNF and trkB. The mean number of BDNF/trkB-IR small/medium-diameter TRG neurons was significantly higher in inflamed rats than in naïve rats. In whole-cell current-clamp experiments, the majority of dissociated small-diameter TRG neurons showed a depolarization response to BDNF that was associated with spike discharge, and the concentration of BDNF that evoked a depolarizing response was significantly lower in the inflamed rats. In addition, the relative number of BDNF-induced spikes during current injection was significantly higher in inflamed rats. The BDNF-induced changes in TRG neuron excitability was abolished by tyrosine kinase inhibitor, K252a. CONCLUSION: The present study provided evidence that BDNF enhances the excitability of the small-diameter TRG neurons projecting onto the Vi/Vc following MM inflammation. These findings suggest that ganglionic BDNF-trkB signaling is a therapeutic target for the treatment of trigeminal inflammatory hyperalgesia.

Suppression of ATP-induced excitability in rat small-diameter trigeminal ganglion neurons by activation of GABAB receptor.

The aim of the present study was to investigate whether a GABAB receptor agonist could modulate ATP-activated neuronal excitability of nociceptive TRG neurons using perforated whole-cell patch-clamp and immunohistochemical techniques. Immunohistochemical analysis revealed that 86% of P2X3 receptor-immunoreactive, small-diameter TRG neurons co-expressed GABAB receptor. Under voltage-clamp conditions (Vh=-60mV), application of ATP activated the inward current in acutely isolated rat TRG neurons in a dose-dependent manner (10-50 µM) and this current could be blocked by pyridoxal-phosphate-6-azophenyl-27,47-disulfonic acid (PPADS) (10 µM), a selective P2 purinoreceptor antagonist. The peak amplitude of ATP-activated currents was significantly inhibited after application of GABAB receptor agonist, baclofen (10-50 µM), in a concentration-dependent and reversible manner. The baclofen-induced inhibition of ATP-activated current was abolished by co-application of 3-amino-2 (4-chlorophenyl)-2hydroxypropysufonic acid) saclofen, a GABAB receptor antagonist (50 µM). Under current-clamp conditions, application of 20 µM ATP significantly depolarized the membrane potential resulting in increased mean action potential frequencies, and these ATP-induced effects were significantly inhibited by baclofen and these effects were antagonized by co-application of saclofen. Together, the results suggested that GABAB receptor activation could inhibit the ATP-induced excitability of small-diameter TRG neurons activated through the P2X3 receptor. Thus, the interaction between P2X3 and GABAB receptors of small-diameter TRG neuronal cell bodies is a potential therapeutic target for the treatment of trigeminal nociception.

Postnatal mandibular cheek tooth development in the miniature pig based on two-dimensional and three-dimensional X-ray analyses.

The miniature pig is a useful large laboratory animal model. Various tissues and organs of miniature pigs are similar to those of humans in terms of developmental, anatomical, immunological, and physiological characteristics. The oral and maxillofacial region of miniature pigs is often used in preclinical studies of regenerative dentistry. However, there is limited information on the dentition and tooth structure of miniature pigs. The purpose of this study was to examine the time-course changes of dentition and tooth structure (especially the root) of the miniature pig mandibular cheek teeth through X-ray analyses using soft X-ray for two-dimensional observations and micro-CT for three-dimensional observations. The mandibles of male Clawn strain miniature pigs (2 weeks and 3, 5, 7, 9, 11, 14, 17, and 29 months of age) were used. X-ray analysis of the dentition of miniature pig cheek teeth showed that the eruption pattern of the miniature pig is diphyodont and that the replacement pattern is vertical. Previous definitions of deciduous and permanent teeth often varied and there has been no consensus on the number of teeth (dentition); however, we found that three molars are present in the deciduous dentition and that four premolars and three molars are present in the permanent dentition. Furthermore, we confirmed the number of tooth roots and root canals. We believe that these findings will be highly useful in future studies using miniature pig teeth.

Isolation and characterization of epithelial and myogenic cells by "fishing" for the morphologically distinct cell types in rat primary periodontal ligament cultures.

The periodontal ligament (PDL) contains various cell populations and plays a central role in the maintenance, repair, and regeneration of the periodontium, i.e., tooth-supporting structures. Because primary cells isolated from PDL tissue are heterogeneous, the establishment of an effective isolation method for cells of interest is desired. In the present study, two morphologically distinct cell types were identified in confluent primary cultures derived from rat PDL. To isolate these cell populations, a small piece of filter paper soaked with trypsin-EDTA was placed directly onto the target cell population, enabling the cells to detach from the culture dish. The filter papers were then transferred into fresh culture dishes to establish outgrowth cultures; these two steps constitute the "cell fishing" method. The "fished" cell types were propagated and subcultured for further analyses. In morphological evaluation, immunocytochemical analyses, and reverse transcription-polymerase chain reaction, the isolated cells exhibited a polygonal appearance or a mono- or multinucleated appearance, with a high cytoplasm-to-nucleus ratio, leading to their being characterized as epithelial or myogenic cell populations, respectively. Surprisingly, a notable proportion of the multinuclear cells in the primary and subsequent isolated cultures demonstrated dramatic, spontaneous contractions, a feature typical of skeletal muscle cells. Finally, the isolated cell populations maintained a normal karyotype with a diploid chromosomal number. These results demonstrated that physiological epithelial and skeletal muscle cells can be obtained from primary PDL cultures without artificial induction using growth factors or chemicals, and can be propagated as individual lineage-committed cell populations; the populations consisted of differentiated and progenitor cells that maintained chromosomal stability. This simple, classical culture procedure provides new insights into the biological properties of PDL cells, which are potentially important for the differentiation of tissue or somatic stem cells and for the development of future cell-based therapies for dental and muscular diseases.

Isolation and characterization of vascular endothelial cells derived from fetal tooth buds of miniature swine.

The aim of the present study was to isolate endothelial cells from tooth buds (unerupted deciduous teeth) of miniature swine. Mandibular molar tooth buds harvested from swine fetuses at fetal days 90-110 were cultured in growth medium supplemented with 15% fetal bovine serum in 100-mm culture dishes until the primary cells outgrown from the tooth buds reached confluence. A morphologically defined set of pavement-shaped primary cells were picked up manually with filter paper containing trypsin/ethylenediamine tetraacetic acid solution and transferred to a separate dish. A characterization of the cellular characteristics and a functional analysis of the cultured cells at passages 3 to 5 were performed using immunofluorescence, a reverse transcriptase polymerase chain reaction assay, a tube formation assay, and transmission electron microscopy. The isolated cells grew in a pavement arrangement and showed the characteristics of contact inhibition upon reaching confluence. The population doubling time was ~48 h at passage 3. As shown by immunocytostaining and western blotting with specific antibodies, the cells produced the endothelial marker proteins such as vascular endothelial cadherin, von Willebrand factor, and vascular endothelial growth factor receptor-2. Observation with time-lapse images showed that small groups of cells aggregated and adhered to each other to form tube-like structures. Moreover, as revealed through transmission electron microscopy, these adherent cells had formed junctional complexes. These endothelial cells from the tooth buds of miniature swine are available as cell lines for studies on tube formation and use in regenerative medical science.

Progression of oral squamous cell carcinoma accompanied with reduced E-cadherin expression but not cadherin switch.

The cadherin switch from E-cadherin to N-cadherin is considered as a hallmark of the epithelial-mesenchymal transition and progression of carcinomas. Although it enhances aggressive behaviors of adenocarcinoma cells, the significance and role of cadherin switch in squamous cell carcinomas (SCCs) are largely controversial. In the present study, we immunohistochemically examined expression of E-cadherin and N-cadherin in oral SCCs (n = 63) and its implications for the disease progression. The E-cadherin-positive carcinoma cells were rapidly decreased at the invasive front. The percentage of carcinoma cells stained E-cadherin at the cell membrane was reduced in parallel with tumor dedifferentiation (P<0.01) and enhanced invasion (P<0.01). In contrast, N-cadherin-positive cells were very limited and did not correlate with the clinicopathological parameters. Mouse tongue tumors xenotransplantated oral SCC cell lines expressing both cadherins in vitro reproduced the reduction of E-cadherin-positive carcinoma cells at the invasive front and the negligible expression of N-cadherin. These results demonstrate that the reduction of E-cadherin-mediated carcinoma cell-cell adhesion at the invasive front, but not the cadherin switch, is an important determinant for oral SCC progression, and suggest that the environments surrounding carcinoma cells largely affect the cadherin expression.

Peripheral inflammation suppresses inward rectifying potassium currents of satellite glial cells in the trigeminal ganglia.

Previous studies indicate that silencing Kir4.1, a specific inward rectifying K(+) (Kir) channel subunit, in sensory ganglionic satellite glial cells (SGCs) induces behavioral hyperalgesia. However, the function of Kir4.1 channels in SGCs in vivo under pathophysiological conditions remains to be determined. The aim of the present study was to examine whether peripheral inflammation in anesthetized rats alters the SGC Kir4.1 current using in vivo patch clamp and immunohistochemical techniques. Inflammation was induced by injection of complete Freund's adjuvant into the whisker pad. The threshold of escape from mechanical stimulation applied to the orofacial area in inflamed rats was significantly lower than in naïve rats. The mean percentage of small/medium diameter trigeminal ganglion (TRG) neurons encircled by Kir4.1-immunoreactive SGCs in inflamed rats was also significantly lower than in naïve rats. In vivo whole-cell recordings were made using SGCs in the trigeminal ganglia (TRGs). Increasing extracellular K(+) concentrations resulted in significantly smaller potentiation of the mean peak amplitude of the Kir current in inflamed compared with naïve rats. In addition, the density of the Ba(2+)-sensitive Kir current associated with small-diameter TRG neurons was significantly lower in inflamed rats compared with naïve rats. Mean membrane potential in inflamed rats was more depolarized than in naïve rats. These results suggest that inflammation could suppress Kir4.1 currents of SGCs in the TRGs and that this impairment of glial potassium homeostasis in the TRGs contributes to trigeminal pain. Therefore, the Kir4.1 channel in SGCs may be a new molecular target for the treatment of trigeminal inflammatory pain.

The effects of oral xylitol administration on bone density in rat femur.

To examine the effects of oral xylitol administration on rat femur bone density, 36 four-week-old male Wistar rats divided into three groups were fed CE-2 diet (control, n = 12) alone or supplemented with 10% (n = 12) or 20% (n = 12) dietary xylitol for 40 days. Biochemical, morphological, and histological analyses were performed. The 10% and 20% xylitol groups showed higher levels of both serum Ca and alkaline phosphatase activity and lower levels of serum tartrate-resistant acid phosphatase than the control group. Although no significant differences in the three-dimensional bone structure or trabecular bone structure of the femur were observed, both xylitol groups showed significantly higher bone density than the control group. Compared to the control group, the 10% and 20% xylitol groups showed an increase in trabeculae. Thus, oral administration of xylitol appears to affect bone metabolism, leading to increased bone density in rat femur.

Novel amelanotic and melanotic cell lines NM78-AM and NM78-MM derived from a human oral malignant melanoma.

Abstract Novel cell lines, designated NM78-AM and NM78-MM, have been established from a malignant melanoma of the cheek oral mucosa. NM78-AM cells were spherical, grew in suspension as clusters, and produced no melanin. In contrast, NM78-MM cells were adherent and produced melanin granules. Initially, NM78-AM cells were grown on fibroblast feeder cells or in growth media supplemented with 10% conditioned medium from fibroblasts, but eventually grew in standard growth media alone. NM78-AM cells had interdigitating microvilli and formed cell clusters. They had large nucleoli, desmosomes, lipid droplets, and well-developed Golgi apparatuses. In contrast, NM78-MM cells grew as adherent neuron-like cells. They had large prominent nucleoli, irregular nuclear membranes, a number of mitochondria, well-developed Golgi apparatuses, melanosomes at various stages of development in the cytoplasm, and the cells secreted melanin granules. Projections from these melanotic cells formed anastomoses with each other. NM78-MM cells stained immunofluorescently for internexin, neuron specific enolase, NF-200, and glial fibrillary acidic protein. These cells were severely aneuploid, approximating to triploidy, and had many marker chromosomes. We used a real-time monitoring system to evaluate oxygen concentrations in culture medium to investigate the susceptibility of both cell lines to various anti-cancer drugs. NM78-AM cells were slightly sensitive to actinomycin D, but not to cisplatin, irinotecan, the irinotecan metabolite SN-38, taxol, taxotere, bleomycin and methotrexate; NM78-MM cells were sensitive to cisplatin, and not to taxol, taxotere, carboplatin, and irinotecan. These new cell lines, NM78-AM and NM78-MM, will be very important for the development of new chemotherapeutics for oral malignant melanoma.

In vivo patch-clamp analysis of response properties of rat primary somatosensory cortical neurons responding to noxious stimulation of the facial skin.

BACKGROUND: Although it has been widely accepted that the primary somatosensory (SI) cortex plays an important role in pain perception, it still remains unclear how the nociceptive mechanisms of synaptic transmission occur at the single neuron level. The aim of the present study was to examine whether noxious stimulation applied to the orofacial area evokes the synaptic response of SI neurons in urethane-anesthetized rats using an in vivo patch-clamp technique. RESULTS: In vivo whole-cell current-clamp recordings were performed in rat SI neurons (layers III-IV). Twenty-seven out of 63 neurons were identified in the mechanical receptive field of the orofacial area (36 neurons showed no receptive field) and they were classified as non-nociceptive (low-threshold mechanoreceptive; 6/27, 22%) and nociceptive neurons. Nociceptive neurons were further divided into wide-dynamic range neurons (3/27, 11%) and nociceptive-specific neurons (18/27, 67%). In the majority of these neurons, a proportion of the excitatory postsynaptic potentials (EPSPs) reached the threshold, and then generated random discharges of action potentials. Noxious mechanical stimuli applied to the receptive field elicited a discharge of action potentials on the barrage of EPSPs. In the case of noxious chemical stimulation applied as mustard oil to the orofacial area, the membrane potential shifted depolarization and the rate of spontaneous discharges gradually increased as did the noxious pinch-evoked discharge rates, which were usually associated with potentiated EPSP amplitudes. CONCLUSIONS: The present study provides evidence that SI neurons in deep layers III-V respond to the temporal summation of EPSPs due to noxious mechanical and chemical stimulation applied to the orofacial area and that these neurons may contribute to the processing of nociceptive information, including hyperalgesia.

Glial cell line-derived neurotrophic factor acutely modulates the excitability of rat small-diameter trigeminal ganglion neurons innervating facial skin.

Glial cell line-derived neurotrophic factor (GDNF) plays an important role in adult sensory neuron function. However, the acute effects of GDNF on primary sensory neuron excitability remain to be elucidated. The aim of the present study was to investigate whether GDNF acutely modulates the excitability of adult rat trigeminal ganglion (TRG) neurons that innervate the facial skin by using perforated-patch clamping, retrograde-labeling and immunohistochemistry techniques. Fluorogold (FG) retrograde labeling was used to identify the TRG neurons innervating the facial skin. The FG-labeled small- and medium-diameter GDNF immunoreactive TRG neurons, and most of these neurons also expressed the GDNF family receptor alpha-1 (GFRalpha-1). In whole-cell voltage-clamp mode, GDNF application significantly inhibited voltage-gated K(+) transient (I(A)) and sustained (I(K)) currents in most dissociated FG-labeled small-diameter TRG neurons. This effect was concentration-dependent and was abolished by co-application of the protein tyrosine kinase inhibitor, K252b. Under current-clamp conditions, the repetitive firing during a depolarizing pulse were significantly increased by GDNF application. GDNF application also increased the duration of the repolarization phase and decreased the duration of the depolarization phase of the action potential, and these characteristic effects were also abolished by co-application of K252b. These results suggest that acute application of GDNF enhances the neuronal excitability of adult rat small-diameter TRG neurons innervating the facial skin, via activation of GDNF-induced intracellular signaling pathway. We therefore conclude that a local release of GDNF from TRG neuronal soma and/or nerve terminals may regulate normal sensory function, including nociception.

Establishment and characterization of the NEYS cell line derived from carcinosarcoma of human ovary with special reference to the susceptibility test of anticancer drugs.

A cell line designated as NEYS was established from ovarian carcinosarcoma (stage IIIc) of a 56-year-old Japanese woman. The extirpated original tumor was carried in growth medium at 0 degrees C to the culture room. The primary culture was done on 20 August 2003. The cell line was composed of angular adhesive cells and showed neoplastic and pleomorphic features, such as bizarre aggregation of chromatin granules, an irregular thickening nuclear membrane and multiple large nucleoli. They grew as multi-layered cultures without contact inhibition. The cells proliferated moderately, and population doubling time was about 56 h. The chromosome number showed an underdiploidy of aneuploidy. The modal chromosome numbers were 37 (36%) and 38 (26%). The cultures produced carcinoembryonic antigen (27.4 ng/mL), carbohydrate antigen 19-9 (210 U/mL), and carbohydrate antigen 125 (526 U/mL). The NEYS cells did not give rise to transplant tumors in nude mice, and showed no susceptibility against cisplatin (CDDP), CPT-11, carboplatin, Paclitaxel, Taxotere and 5-FU. This cell line is useful for studies on the histogenesis of carcinosarcoma and susceptibility of cancer drugs in human ovarian carcinosarcoma. The immunohistochemical and ultrastructual analysis demonstrated that NEYS cells showed epithelial and mesenchymal differentiation, and supported the metaplasis theory as the cause of carcinosarcoma.

Effects of amifostine administration prior to irradiation to the submandibular gland in mice: autoradiographic study using 3H-leucine.

We histochemically investigated the radioprotection of organelle in acinus on the submandibular gland using amifostine. Mice were divided into those without amifostine administration or irradiation (group A), with amifostine administration and without irradiation (group B), without amifostine and with 5 Gy of irradiation (group C), and with amifostine and 5 Gy of irradiation (group D). In groups B and D were given 100 mg/kg of amifostine at 30 minutes prior to irradiation to the area equivalent to the submandibular gland. HE staining revealed that group D showed atrophy of the acinar cells, though the change was milder than that seen in group C. In AZAN-Mallory staining, fibrogenesis were found more often in group C as compared to the other groups. In Light Microscopic Autoradiography findings, the ratio of reduced silver particles in the acinar cells was lower in group C than in the other groups at 30 minutes after RI administration. Microstructure findings showed the findings were very similar to those of group A. In Electron microscopic Autoradiography, the ratio among the secretory granules was lower in group C at 120 minutes after RI administration. Our results indicate that amifostine can alleviate xerostomia caused by late effect from radiation.

Temporomandibular joint inflammation decreases the voltage-gated K+ channel subtype 1.4-immunoreactivity of trigeminal ganglion neurons in rats.

Voltage-gated K+ (Kv) channels are one of the important physiological regulators of the membrane potentials in excitable cells, including sensory ganglion neurons. The aim of the present study was to investigate whether temporomandibular joint (TMJ) inflammation alters expression of Kv channel subtype 1.4 (Kv1.4) of trigeminal ganglion (TRG) neurons innervating TMJ relating allodynia (pain caused by normally innoxious stimulation), by using both behavioral and immunohistochemical techniques. TMJ inflammation was induced by injection of Complete Freund's Adjuvant (CFA) into the rat TMJ. The threshold for escape from mechanical stimulation applied to the orofacial area in TMJ inflamed rats was significantly lower than that in naïve rats. TMJ afferents were identified by fluorogold (FG) labeling. The mean numbers of Kv1.4-/neurofilament (NF) 200(myelinated fiber marker) positive- and negative-immunoreactivities FG-labeled small-/medium-diameter TRG neurons in inflamed rats were significantly decreased when compared with those in the naïve rats. These findings suggest that TMJ inflammation reduces the expression of Kv1.4 subunits in the small-/medium sized (Adelta-/C-) TRG neurons and this may contribute to trigeminal inflammatory allodynia in TMJ disorder. These results lead us to suggest that Kv channel openers may be a potential therapeutic agents for prevention of mechanical allodynia.

Allopurinol gel mitigates radiation-induced mucositis and dermatitis.

It has not been verified whether allopurinol application is beneficial in decreasing the severity of radiation-induced oral mucositis and dermatitis. Rats were divided into 4 groups and received 15 Gy irradiation on the left whisker pad. Group 1 received only irradiation. Group 2 was maintained by applying allopurinol/carrageenan-mixed gel (allopurinol gel) continuously from 2 days before to 20 days after irradiation. Group 3 had allopurinol gel applied for 20 days after radiation. Group 4 was maintained by applying carrageenan gel continuously from 2 days before to 20 days after irradiation. The intra oral mucosal and acute skin reactions were assessed daily using mucositis and skin score systems. The escape thresholds for mechanical stimulation to the left whisker pad were measured daily. In addition, the irradiated tissues at the endpoint of this study were compared with naïve tissue. Escape threshold in group 2 was significantly higher than that in group 1, and mucositis and skin scores were much improved compared with those of group 1. Concerning escape threshold, mucositis and skin scores in group 3 began to improve 10 days after irradiation. Group 4 showed severe symptoms of mucositis and dermatitis to the same extent as that observed in group 1. In the histophathological study, the tissues of group 1 showed severe inflammatory reactions, compared with those of group 2. These results suggest that allopurinol gel application can mitigate inflammation reactions associated with radiation-induced oral mucositis and dermatitis.

Enhanced excitability of nociceptive trigeminal ganglion neurons by satellite glial cytokine following peripheral inflammation.

Peripheral nerve injury activates satellite cells to produce interleukin 1beta (IL-1beta) which mediates inflammation and hyperalgesia. This study investigated the hypothesis that activation of satellite glial cells modulates the excitability of trigeminal ganglion (TRG) neurons via IL-1beta following inflammation. Inflammation was induced by injection of complete Freund's adjuvant (CFA) into the whisker pad area. The threshold for escape from mechanical stimulation applied to the whisker pad in inflamed rats was significantly lower than that in control. Two days post-CFA injection, the mean percentage of TRG neurons encircled by glial fibrillary acidic protein (GFAP)-/IL-1beta-immunoreactive cells was significantly increased compared to controls. GFAP and IL-1beta immunoreactivities were coexpressed in the same cells. Fluorogold (FG) labeling identified the site of inflammation. The number of FG-labeled IL-receptor type I (IL-1RI) TRG neurons in inflamed rats was significantly greater than in controls. In FG-labeled small TRG neurons, the size of IL-1beta (1 nM) induced-depolarization in inflamed rats was larger than in controls. IL-1beta application significantly increased firing rates evoked by depolarizing pulses in the neurons of inflamed rats, compared to controls. The response to IL-1beta was abolished by treatment with the IL-1RI antagonist. These results suggest that activation of satellite glial cells modulates the excitability of small-diameter TRG neurons via IL-1beta following inflammation, and that the upregulation of IL-1RI in the soma may contribute to the mechanism underlying inflammatory hyperalgesia. Therefore IL-1beta blockers are potential therapeutic agents for prevention of trigeminal hyperalgesia.