Bactericidal and cytotoxic effects of acid-electrolyzed functional water.

Sodium hypochlorite (NaOCl) is widely used as an antimicrobial irrigant; however, it has cytotoxic and neurotoxic effects. For these reasons, development of new, safe irrigants other than NaOCl is long overdue. In the present study, the antimicrobial and noxious effects of acid-electrolyzed functional water (FW) were evaluated and compared with those of NaOCl. Enterococcus faecalis, Streptococcus mutans, Porphyromonas gingivalis, or Candida albicans were mixed with each tested solution for 30 s. The mixtures were then plated on brain-heart infusion agar plates, after which colony numbers were counted. Serially diluted acid FW was used to determine the actual chloride concentration (ACC) required for a bactericidal effect. Noxious effects were evaluated by measuring lactate dehydrogenase released from HeLa cells. Acid FW and NaOCl had similar bactericidal effects against all bacterial species but not against C. albicans. An ACC of at least 10 ppm was required in order to ensure effective bacteriocidal activity and induce significant lactate dehydrogenase release. Acid FW-treated HeLa cells exhibited healthy growth, with slight retardation as compared with non-treated cells. Because of its efficient bactericidal, and less noxious, effects on human cells, acid FW may be a useful irrigant for effective root canal treatment.

Singlet oxygen generated by a new nonthermal atmospheric pressure air plasma device exerts a bactericidal effect on oral pathogens.

Oral diseases generally have certain bacteria associated with them. Non-thermal atmospheric pressure plasma (NTAP), generated at atmospheric pressure and room temperature, incorporates several molecules, including reactive oxygen species, that can inactivate various bacteria including oral pathogens. For this reason, several NTAP devices have been developed to treat oral diseases. Use of noble gases can enhance the bactericidal efficacy of NTAP, but this requires additional gas supply equipment. Therefore, a new NTAP device that employs ambient air as the working gas was developed. The device generates non-thermal atmospheric pressure air plasma. Here, the singlet oxygen (1O2) levels generated, their bactericidal effects on oral pathogens (Streptococcus mutans, Porphyromonas gingivalis, and Enterococcus faecalis), and the bacterial oxidative stress they imposed were measured. 1O2 generation in phosphatebuffered saline was assessed qualitatively using electron spin resonance (ESR) spectroscopy, and bactericidal efficacy was evaluated by counting of colony-forming units/mL. Bacterial oxidative stress was determined by measurement of hydrogen peroxide (H2O2) and superoxide dismutase (SOD) activity. ESR indicated that the level of 1O2 increased significantly and time-dependently, and was inversely correlated with distance, but the bactericidal effects were correlated only with treatment time (not distance) as H2O2 increased and SOD levels decreased, suggesting that the new device has potential applicability for treatment of oral disease.

Extracellular glucose-dependent IPSC enhancement by leptin in fast-spiking to pyramidal neuron connections via JAK2-PI3K pathway in the rat insular cortex.

Leptin is produced in the adipocytes and plays a pivotal role in regulation of energy balance by controlling appetite and metabolism. Leptin receptors are widely distributed in the brain, especially in the hypothalamus, hippocampus, and neocortex. The insular cortex (IC) processes gustatory and visceral information, which functionally correlate to feeding behavior. However, it is still an open issue whether and how leptin modulates IC neural activities. Our paired whole-cell patch-clamp recordings using IC slice preparations demonstrated that unitary inhibitory postsynaptic currents (uIPSCs) but not uEPSCs were potentiated by leptin in the connections between pyramidal (PNs) and fast-spiking neurons (FSNs). The leptin-induced increase in uIPSC amplitude was accompanied by a decrease in paired-pulse ratio. Under application of inhibitors of JAK2-PI3K but not MAPK pathway, leptin did not change uIPSC amplitude. Variance-mean analysis revealed that leptin increased the release probability but not the quantal size and the number of release site. These electrophysiological findings suggest that the leptin-induced uIPSC increase is mediated by activation of JAK2-PI3K pathway in presynaptic FSNs. An in vivo optical imaging revealed that leptin application decreased excitatory propagation in IC induced by electrical stimulation of IC. These leptin-induced effects were not observed under the low energy states: low glucose concentration (2.5 mM) in vitro and one-day-fasting condition in vivo. However, leptin enhanced uIPSCs under application of low glucose with an AMPK inhibitor. These results suggest that leptin suppresses IC excitation by facilitating GABA release in FSN→PN connections, which may not occur under a hunger state.

Connexin 43 expression in satellite glial cells contributes to ectopic tooth-pulp pain.

Pulpitis often causes referred pain in opposing teeth. However, the precise mechanism underlying ectopic pain associated with tooth-pulp inflammation remains unclear. We performed the present study to test the hypothesis that functional interactions between satellite glial cells (SGCs) and trigeminal ganglion (TG) neurons are involved in ectopic orofacial pain associated with tooth-pulp inflammation. Digastric muscle electromyograph (D-EMG) activity elicited by administration of capsaicin into the upper second molar pulp (U2) was analyzed to evaluate noxious reflex responses. D-EMG activity was significantly increased in rats with lower first molar (L1) inflammation relative to saline-treated rats. Significantly increased expression of glial fibrillary acid protein (GFAP), a marker of activated glial cells, and connexin 43 (Cx43), a gap-junction protein, was observed in activated SGCs surrounding U2-innervating TG-neurons after L1-pulp inflammation. Daily administration of Gap26, a Cx43-inhibiting mimetic peptide, into the TG significantly suppressed capsaicin-induced D-EMG activity enhancement and reduced the percentage of fluorogold-labeled (U2-innervated) cells that were surrounded by GFAP-immunoreactive (IR) and Cx43-IR cells after L1-pulp inflammation. These findings indicate that tooth-pulp inflammation induces SGC activation and subsequent spread of SGC activation in the TG via Cx43-containing gap junctions. Thus, remote neuron excitability becomes enhanced in the TG following tooth-pulp inflammation, resulting in ectopic tooth-pulp pain in the contralateral tooth.

Expression of the Forkhead box transcription factor Foxo3a in human periapical granulomas.

It has been reported that Forkhead box transcription factor class O3a (Foxo3a) is expressed in rheumatoid arthritis, a chronic inflammatory condition accompanied by bone resorption, and plays a role in its pathology. However, it has remained unclear whether Foxo3a is involved in the pathogenesis of periapical granulomas. The present study was performed to compare the expression of Foxo3a in periapical granulomas and healthy gingival tissues. Samples were obtained surgically from patients, and subjected to hematoxylin-eosin staining for histopathologic diagnosis. Two-color immunofluorescence staining was also performed using antibodies against Foxo3a and markers for three types of inflammatory cells: neutrophils, T lymphocytes, and B lymphocytes. This revealed that Foxo3a was expressed in all three cell types in periapical granulomas but not in healthy gingival tissues. Foxo3a was expressed in 82.1%, 78.3%, and 77.5% of neutrophils, T lymphocytes, and B lymphocytes, respectively, and statistical analysis using the Kruskal-Wallis test followed by the Steel-Dwass test showed no significant difference of Foxo3a expression among the three cell types. Our results suggest that Foxo3a transcription factors may be involved in the pathogenesis of periapical granulomas.

Expression of silent information regulator 2 homolog 1 (SIRT1) in periapical granulomas.

Silent information regulator 2 homolog 1 (SIRT1) inhibits oxidative injury and has anti-inflammatory effects. SIRT1 may be involved in healing of periapical periodontitis; however, SIRT1 expression in periapical periodontitis lesions has not been investigated. This study evaluated SIRT1 expression and a marker of oxidative stress-8-hydroxy-2'-deoxyguanosine (8-OHdG)-in periapical granulomas. First, we used real-time polymerase chain reaction to determine whether U-937 monocytes express SIRT1. U-937 cells treated with the SIRT1 activator resveratrol exhibited the highest SIRT1 mRNA level after 6-h incubation. By contrast, treating cells with the SIRT1 inhibitor sirtinol returned SIRT1 expression level to that of the control. In addition, immunocytochemical analysis using cytospin specimens showed that U-937 cells co-expressed SIRT1 and Ki-67. Dual-color immunofluorescence imaging showed that round cells in periapical granulomas co-expressed SIRT1 and 8-OHdG; however, neither was expressed in healthy gingival tissues. The number of 8-OHdG-expressing cells was significantly greater than the number of SIRT1-expressing cells. Our findings suggest that macrophages express SIRT1 and that wound healing in periapical granulomas is enhanced by a SIRT1-mediated reduction in the level of oxidative stress.

Microcomputed tomographic evaluation of techniques for warm gutta-percha obturation.

Transparent epoxy resin root canal models were used to evaluate vertical condensation techniques for obturating lateral canals. The root canal model was configured with a straight main root canal and four right-angled lateral canals at 1.0 and 3.0 mm from the apex. Root canal obturation was performed with Thermafil, Obtura II, or NT condenser. Obturation volume in lateral canals was measured by three-dimensional microcomputed tomography, and one-way analysis of variance was used to analyze differences between groups. Lateral canals at 1.0 and 3.0 mm were uniformly filled by all obturation methods. Among the three obturation methods, Thermafil resulted in the highest obturation volumes for all lateral canals.

Role of medullary astroglial glutamine synthesis in tooth pulp hypersensitivity associated with frequent masseter muscle contraction.

Background The mechanisms underlying tooth pulp hypersensitivity associated with masseter muscle hyperalgesia remain largely underinvestigated. In the present study, we aimed to determine whether masseter muscle contraction induced by daily electrical stimulation influences the mechanical head-withdrawal threshold and genioglossus electromyography activity caused by the application of capsaicin to the upper first molar tooth pulp. We further investigated whether astroglial glutamine synthesis is involved in first molar tooth pulp hypersensitivity associated with masseter muscle contraction. Methods The first molar tooth pulp was treated with capsaicin or vehicle in masseter muscle contraction or sham rats, following which the astroglial glutamine synthetase inhibitor methionine sulfoximine or Phosphate buffered saline (PBS) was applied. Astroglial activation was assessed via immunohistochemistry. Results The mechanical head-withdrawal threshold of the ipsilateral masseter muscle was significantly decreased in masseter muscle contraction rats than in sham rats. Genioglossus electromyography activity was significantly higher in masseter muscle contraction rats than sham rats. Glial fibrillary acidic protein-immunoreactive cell density was significantly higher in masseter muscle contraction rats than in sham rats. Administration of methionine sulfoximine induced no significant changes in the density of glial fibrillary acidic protein-immunoreactive cells relative to PBS treatment. However, mechanical head-withdrawal threshold was significantly higher in masseter muscle contraction rats than PBS-treated rats after methionine sulfoximine administration. Genioglossus electromyography activity following first molar tooth pulp capsaicin treatment was significantly lower in methionine sulfoximine-treated rats than in PBS-treated rats. In the ipsilateral region, the total number of phosphorylated extracellular signal-regulated protein kinase immunoreactive cells in the medullary dorsal horn was significantly smaller upon first molar tooth pulp capsaicin application in methionine sulfoximine-treated rats than in PBS-treated rats. Conclusions Our results suggest that masseter muscle contraction induces astroglial activation, and that this activation spreads from caudal to the obex in the medullary dorsal horn, resulting in enhanced neuronal excitability associated with astroglial glutamine synthesis in medullary dorsal horn neurons receiving inputs from the tooth pulp. These findings provide significant insight into the mechanisms underlying tooth pulp hypersensitivity associated with masseter muscle contraction.

Involvement of transient receptor potential vanilloid 1 channel expression in orofacial cutaneous hypersensitivity following tooth pulp inflammation.

A study was conducted to evaluate the mechanisms underlying ectopic orofacial pain associated with tooth pulp inflammation in rats. We observed a significant decrease in the head withdrawal threshold (HWT) response to mechanical and heat stimuli applied to the ipsilateral facial skin upon application of complete Freund's adjuvant (CFA) to the upper first molar (M1TP) in comparison to application of vehicle. A large number of trigeminal ganglion (TG) neurons showed transient receptor potential vanilloid 1 (TRPV1) immunoreactivity (IR), and some of them were retrogradely labeled with fluorogold injected into the facial skin. A large number of cells showing IR for glial fibrillary acidic protein (GFAP) were observed in the 2nd compared to the 1st or 3rd branch regions of the TG, and TG cells innervating the facial skin were also surrounded by GFAP-IR cells. After administration of TRPV1 antagonist into the facial skin of M1TP CFA-treated rats, the decrease of HWTs in response to mechanical and heat stimulation of the facial skin was significantly reversed. The present findings suggest that the excitability of TG neurons is enhanced upon tooth pulp inflammation, leading to overexpression of TRPV1 in TG neurons innervating the facial skin, and that satellite glial cells are also activated, resulting in the development of ectopic orofacial pain.

Ablation of C-fibers decreases quantal size of GABAergic synaptic transmission in the insular cortex.

The primary sensory cortex exhibits neuroplastic changes responding to sensory disturbances, and GABAergic synaptic transmission plays a critical role in the regulation of plasticity. The insular cortex (IC) integrates orofacial nociceptive signals conveyed via myelinated Aδ- and unmyelinated C-fibers. However, it has been unknown whether a disturbance of nociceptive inputs, such as a deletion of the peripheral nerves, alters GABAergic local circuit in IC. The present study elucidated GABAergic synaptic transmission in the model rat whose C-fibers were ablated by capsaicin injection 1-2 days after birth. In vivo optical imaging revealed that capsaicin-treated rats showed a facilitative excitatory propagation in IC responding to dental pulp stimulation. Whole-cell patch-clamp recording from pyramidal neurons (Pyr) demonstrated that capsaicin-treated rats showed the smaller amplitude of miniature inhibitory postsynaptic currents (IPSCs) than sham-treated rats without changing the frequency. Furthermore, replacement of extracellular Ca2+ to Sr2+, which induces an asynchronous release of neurotransmitters in the quantal size, induced a smaller amplitude of asynchronous unitary IPSCs recorded from fast-spiking GABAergic interneuron to Pyr connections in capsaicin-treated rats than sham-treated rats. These results suggest that capsaicin treatment depresses IPSCs via a postsynaptic mechanism. To confirm this possibility, the variance-mean analysis of unitary IPSCs was employed and we found that quantal size of GABAergic synaptic transmission was smaller in capsaicin-treated rats than in sham-treated rats. These results suggest that ablation of C-fibers induces plastic changes in GABAergic synaptic transmission by decreasing postsynaptic GABAA receptor-mediated conductance, which is a possible mechanism of the facilitative excitation in IC of capsaicin-treated rats.

Involvement of the calcium-sensing receptor in mineral trioxide aggregate-induced osteogenic gene expression in murine MC3T3-E1 cells.

Mineral trioxide aggregate (MTA) has excellent biocompatibility as well as bioactivity, including an ability to induce osteoblast differentiation. We examined the effects of the calcium-sensing receptor (CaSR) on osteogenic gene expression induced by MTA. MC3T3-E1 cells were cultured with or without (control) MTA. The expression levels of Runx2, type I collagen, and CaSR genes were analyzed by real-time polymerase chain reaction and their products were measured using enzyme-linked immunosorbent assays. The levels were increased significantly in cells exposed to MTA compared with control. Next, MC3T3-E1 cells were cultured with MTA and EGTA (a calcium chelator), because calcium ions were released continuously from MTA into the culture. Expression levels were decreased to control levels by MTA plus EGTA. NPS2143 (a CaSR antagonist) also reduced MTA-induced gene expression. These results suggest that MTA induced osteogenic gene expressions of Runx2 and type I collagen via CaSR in MC3T3-E1 cells.

Bactericidal effect of hydroxyl radicals generated by the sonolysis and photolysis of hydrogen peroxide for endodontic applications.

The aim of endodontic root canal treatment is the elimination of bacteria and their products from an infected tooth root canal. To effectively disinfect a root canal, an ultrasonic irrigation system, in which hydroxyl radicals (HO·) generated artificially by sonolysis of H2O2, was developed previously for endodontic applications and was demonstrated to have bactericidal efficacy against Enterococcus faecalis. To improve this system, we examined the in vitro bactericidal effects of HO· generated from H2O2, activated by simultaneous irradiation with ultrasound for sonolysis and dental LED light for photolysis with a peak wavelength of 405 nm. Regarding the LED irradiation, two methods were used: (i) 'ideal' experimental conditions (irradiation close to the glass tube), and (ii) simulated endodontic conditions (more distant irradiation of a masked glass tube). In these conditions, HO· generation from H2O2 was detected by electron spin resonance (ESR) spectroscopy, and bactericidal efficacy against E. faecalis was assessed by measuring the colony forming units (CFU)/mL. The results indicated that HO· generation by ESR measurements and the bactericidal effect on E. faecalis by viable count using CFU/mL were enhanced significantly in a time-dependent manner in both conditions. In a comparison of these conditions, bactericidal activity under 'ideal' experimental conditions was similar to that under simulated endodontic conditions. Moreover, the irradiation time for effective killing of E. faecalis through the sonolysis and photolysis of H2O2 under simulated endodontic conditions was shorter than that with sonolysis alone. These results demonstrate that H2O2 activated by ultrasound and LED light may be a safe and effective disinfection technique for endodontic root canal treatment.

Evaluation of Qualitative Changes in Simulated Periodontal Ligament and Alveolar Bone Using a Noncontact Electromagnetic Vibration Device with a Laser Displacement Sensor.

Evaluating periodontal tissue condition is an important diagnostic parameter in periodontal disease. Noncontact electromagnetic vibration device (NEVD) was previously developed to monitor this condition using mechanical parameters. However, this system requires accelerometer on the target tooth. This study assessed application of laser displacement sensor (LDS) to NEVD without accelerometer using experimental tooth models. Tooth models consisted of cylindrical rod, a tissue conditioner, and polyurethane or polyurethane foam to simulate tooth, periodontal ligament, and alveolar bone, respectively. Tissue conditioner was prepared by mixing various volumes of liquid with powder. Mechanical parameters (resonant frequency, elastic modulus, and coefficient of viscosity) were assessed using NEVD with the following methods: Group A, measurement with accelerometer; Group B, measurement with LDS in the presence of accelerometer; and Group C, measurement with LDS in the absence of accelerometer. Mechanical parameters significantly decreased with increasing liquid volume. Significant differences were also observed between the polyurethane and polyurethane foam models. Meanwhile, no statistically significant differences were observed between Groups A and B; however, most mechanical parameters in Group C were significantly larger and more distinguishable than those of Groups A and B. LDS could measure mechanical parameters more accurately and clearly distinguished the different periodontal ligament and alveolar bone conditions.

Use of a laser displacement sensor with a non-contact electromagnetic vibration device for assessment of simulated periodontal tissue conditions.

A non-contact electromagnetic vibration device (NEVD) was previously developed to monitor the condition of periodontal tissues by assessing mechanical parameters. This system requires placement of an accelerometer on the target tooth, to detect vibration. Using experimental tooth models, we evaluated the performance of an NEVD system with a laser displacement sensor (LDS), which does not need an accelerometer. Simulated teeth (polyacetal rods) were submerged at various depths in simulated bone (polyurethane or polyurethane foam) containing simulated periodontal ligament (tissue conditioner). Then, mechanical parameters (resonant frequency, elastic modulus, and viscosity coefficient) were assessed using the NEVD with the following detection methods: Group 1, measurement with an accelerometer; Group 2, measurement with an LDS in the presence of the accelerometer; and Group 3, measurement with an LDS in the absence of the accelerometer. Statistical analyses were performed using nonparametric methods (n = 5) (P < 0.05). The three mechanical parameters significantly increased with increasing depth. In addition, the mechanical parameters significantly differed between the polyurethane and polyurethane foam models. Although Groups 1 and 2 did not significantly differ, most all mechanical parameters in Group 3 were significantly larger and more distinguishable than those in Groups 1 and 2. The LDS was more accurate in measuring mechanical parameters and better able to differentiate periodontal tissue conditions. (J Oral Sci 58, 93-99, 2016).

Developmental disturbance of a mandibular central incisor following trauma to the primary predecessor.

When a primary tooth sustains a traumatic injury, development of the permanent tooth can be disturbed, leading to various malformations. This case report details the 7-year follow-up of a developmental anomaly of a central mandibular incisor in a 10-year-old Japanese girl with a history of dental trauma that had occurred at age 4. The trauma had resulted in unusual crown dilaceration in the permanent successor, which exhibited a discoloured and abnormal crown morphology. Radiographic examination revealed crown dilaceration of the tooth, which had a curved root canal.

Epstein-Barr virus infection in chronically inflamed periapical granulomas.

Periapical granulomas are lesions around the apex of a tooth caused by a polymicrobial infection. Treatment with antibacterial agents is normally performed to eliminate bacteria from root canals; however, loss of the supporting alveolar bone is typically observed, and tooth extraction is often selected if root canal treatment does not work well. Therefore, bacteria and other microorganisms could be involved in this disease. To understand the pathogenesis of periapical granulomas more precisely, we focused on the association with Epstein-Barr virus (EBV) using surgically removed periapical granulomas (n = 32). EBV DNA was detected in 25 of 32 periapical granulomas (78.1%) by real-time PCR, and the median number of EBV DNA copies was approximately 8,688.01/µg total DNA. In contrast, EBV DNA was not detected in healthy gingival tissues (n = 10); the difference was statistically significant according to the Mann-Whitney U test (p = 0.0001). Paraffin sections were also analyzed by in situ hybridization to detect EBV-encoded small RNA (EBER)-expressing cells. EBER was detected in the cytoplasm and nuclei of B cells and plasma cells in six of nine periapical granulomas, but not in healthy gingival tissues. In addition, immunohistochemical analysis for latent membrane protein 1 (LMP-1) of EBV using serial tissue sections showed that LMP-1-expressing cells were localized to the same areas as EBER-expressing cells. These data suggest that B cells and plasma cells in inflamed granulomas are a major source of EBV infection, and that EBV could play a pivotal role in controlling immune cell responses in periapical granulomas.

Effect of a novel fluorapatite-forming calcium phosphate cement with calcium silicate on osteoblasts in comparison with mineral trioxide aggregate.

We compared the effects of treatment with fluorapatite-forming calcium phosphate cement (FA-forming CPC) containing tricalcium silicate (TCS) and those of mineral trioxide aggregate (MTA), the gold standard endodontic cement, on cultured osteoblast-like cells (ROS 17/2.8 cells; ROS cells). The FA-forming CPC powder consisted of 61.29% CaHPO4, 32.26% CaCO3, and 6.45% NaF. One part TCS was combined with nine parts FA-forming CPC powder to make FA-forming CPC with TCS. A 1.5-M phosphate solution was mixed as a cement liquid with a powder/liquid ratio of 2.22. Cell culture was carried out using cell culture inserts, whereby each test material was put on a porous membrane insert in the cell culture plate. Proliferation, morphologic changes, and alkaline phosphatase activity in ROS cells were measured in the presence of FA-forming CPC with TCS and MTA and compared. The logarithmic growth phase and cellular morphologic changes in ROS cells were identical in all experimental groups. Additionally, no significant difference in alkaline phosphatase activity was noted in ROS cells exposed to FA-forming CPC with TCS and those exposed to MTA. In conclusion, FA-forming CPC with TCS has characteristics identical to those of MTA under the present experimental conditions and may thus be useful for endodontic applications.

Development of a novel fluorapatite-forming calcium phosphate cement with calcium silicate: in vitro and in vivo characteristics.

Aim of this study was to develop a novel fluorapatite-forming calcium phosphate cement (FA-CPC) with tricalcium silicate (TCS) for endodontic applications and to examine its in vitro and in vivo characteristics. The FA-CPC powder consisted of 62.8% CaHPO4, 30.8% CaCO3, and 6.4% NaF. One part of TCS was combined with 9 parts of FA-CPC powder (FA-CPC with TCS). A 1.5 M phosphate solution was used as cement liquid. Setting time (ST), diametral tensile strength (DTS), phase composition by X-ray diffraction (XRD), and cement alkalinity were analyzed. Cement biocompatibility was assessed using rat subcutaneous model. Cement ST was 10.3±0.6 min and DTS was 3.89±0.76 MPa. XRD patterns showed that highly crystalline apatitic material was the only significant phase present and pH value was approximate 11.0. FA-CPC with TCS demonstrated similar biocompatibility as that of mineral trioxide aggregate control. These results suggest that FA-CPC with TCS may be useful for endodontic applications.

Involvement of trigeminal transition zone and laminated subnucleus caudalis in masseter muscle hypersensitivity associated with tooth inflammation.

A rat model of pulpitis/periapical periodontitis was used to study mechanisms underlying extraterritorial enhancement of masseter response associated with tooth inflammation. Periapical bone loss gradually increased and peaked at 6 weeks after complete Freund's adjuvant (CFA) application to the upper molar tooth pulp (M1). On day 3, the number of Fos-immunoreactive (IR) cells was significantly larger in M1 CFA rats compared with M1 vehicle (veh) rats in the trigeminal subnucleus interpolaris/caudalis transition zone (Vi/Vc). The number of Fos-IR cells was significantly larger in M1 CFA and masseter (Mass) capsaicin applied (M1 CFA/Mass cap) rats compared with M1 veh/Mass veh rats in the contralateral Vc and Vi/Vc. The number of phosphorylated extracellular signal-regulated kinase (pERK)-IR cells was significantly larger in M1 CFA/Mass cap and M1 veh/Mass cap rats compared to Mass-vehicle applied rats with M1 vehicle or CFA in the Vi/Vc. Pulpal CFA application caused significant increase in the number of Fos-IR cells in the Vi/Vc but not Vc on week 6. The number of pERK-IR cells was significantly lager in the rats with capsaicin application to the Mass compared to Mass-vehicle treated rats after pulpal CFA- or vehicle-application. However, capsaicin application to the Mass did not further affect the number of Fos-IR cells in the Vi/Vc in pulpal CFA-applied rats. The digastric electromyographic (d-EMG) activity after Mass-capsaicin application was significantly increased on day 3 and lasted longer at 6 weeks after pulpal CFA application, and these increase and duration were significantly attenuated by i.t. PD98059, a MEK1 inhibitor. These findings suggest that Vi/Vc and Vc neuronal excitation is involved in the facilitation of extraterritorial hyperalgesia for Mass primed with periapical periodontitis or acute pulpal-inflammation. Furthermore, phosphorylation of ERK in the Vi/Vc and Vc play pivotal roles in masseter hyperalgesia after pulpitis or periapical periodontitis.

Bactericidal effect of hydroxyl radicals generated from a low concentration hydrogen peroxide with ultrasound in endodontic treatment.

One approach to enhance the disinfection of root canals in endodontic treatment is ultrasonic irrigation with sodium hypochlorite. Reactive oxygen species, such as hydroxyl radical, are generated by biological defense systems to kill invading bacteria. Ultrasonic irrigation with hydrogen peroxide may be a promising option to increase hydroxyl radical generation. We examined the bactericidal effects of hydroxyl radical generated from low concentration hydrogen peroxide with ultrasound in vitro. An ultrasonic tip was submerged in 0.5 or 1.0 M hydrogen peroxide in a microfuge tube. hydrogen peroxide was irradiated with the ultrasound, the tip of which was maintained centered in the tube to mimic ultrasonic irrigation. Hydroxyl radical generation was assessed by electron spin resonance spectroscopy. Subsequently, Enterococcus faecalis suspension in hydrogen peroxide was prepared and irradiated as described above. Bactericidal effects were assessed by viable counting. Electron spin resonance measurements showed that hydroxyl radical generation increased significantly in a time- and dose-dependent manner (two-way analysis of variance and Tukey's test, p<0.05). Moreover, the bactericidal effects of hydrogen peroxide against Enterococcus faecalis were enhanced by ultrasonic irradiation in a time- and dose-dependent manner. These results suggest that ultrasonic irrigation in the presence of low concentration hydrogen peroxide can serve as a disinfection strategy in endodontic treatment.