Bone formation and deposition are initiated by sensory nerve infiltration in adaptive bone remodeling. Here, we focused on the role of Semaphorin 3A (Sema3A), expressed by sensory nerves, in mechanical loads-induced bone formation and nerve withdrawal using orthodontic tooth movement (OTM) model. Firstly, bone formation was activated after the 3rd day of OTM, coinciding with a decrease in sensory nerves and an increase in pain threshold. Sema3A, rather than nerve growth factor (NGF), highly expressed in both trigeminal ganglion and the axons of periodontal ligament following the 3rd day of OTM. Moreover, in vitro mechanical loads upregulated Sema3A in neurons instead of in human periodontal ligament cells (hPDLCs) within 24 hours. Furthermore, exogenous Sema3A restored the suppressed alveolar bone formation and the osteogenic differentiation of hPDLCs induced by mechanical overload. Mechanistically, Sema3A prevented overstretching of F-actin induced by mechanical overload through ROCK2 pathway, maintaining mitochondrial dynamics as mitochondrial fusion. Therefore, Sema3A exhibits dual therapeutic effects in mechanical loads-induced bone formation, both as a pain-sensitive analgesic and a positive regulator for bone formation.
Humans ; Bone Remodeling ; Cell Differentiation ; Osteogenesis ; Semaphorin-3A/pharmacology* ; Trigeminal Ganglion/metabolism*

The purpose of this study was to establish a model of trigeminal neuralgia (TN) through an approach from lower edge of cheekbone and to observe the functional changes in the voltage-gated potassium currents in the cultured trigeminal ganglion (TG) neurons. Thirty Sprague-Dawley male rats were divided into two groups, the sham-operated (sham) group and the operated group. The TN model was carried out by using a chronic constriction injury of the infraorbital nerve (ION-CCI) from lower edge of cheekbone. Peripheral pain threshold test and whole-cell patch clamp recording were used to determine the difference between sham and ION-CCI rats. The withdrawal threshold of whisker pad in operated side of ION-CCI rat was decreased significantly from 6 d after operation and then maintained until 21 d, with the lowest on the 15th day. The threshold of whisker pad in non-operated side of operated rats was also decreased significantly compared with that in the sham group. Delayed rectifier potassium current (I(K)) in cultured ION-CCI TG neurons was decreased significantly compared with that in the sham group. Transient outward potassium currents (I(A)) in both operated and non-operated sides of TG neurons from ION-CCI rats were also reduced significantly compared with that in the sham group. The present study provided a new method of ION-CCI. In this model, the decrease of I(A) and I(K) might contribute, at least in part, to the decrease in mechanical pain threshold of whisker pad and the subsequent hyperalgia.
Animals ; Cells, Cultured ; Constriction ; Disease Models, Animal ; Hyperalgesia ; Male ; Pain Threshold ; Patch-Clamp Techniques ; Potassium Channels ; metabolism ; Rats ; Rats, Sprague-Dawley ; Trigeminal Ganglion ; metabolism ; Trigeminal Neuralgia ; physiopathology ; Vibrissae

BACKGROUNDAlthough flunarizine has been widely used for migraine prophylaxis with clear success, the mechanisms of its actions in migraine prophylaxis are not completely understood. The aim of this study was to investigate the effects of flunarizine on tetrodotoxin-resistant Na(+) channels and high-voltage activated Ca(2+) channels of acutely isolated mouse trigeminal ganglion neurons.

METHODSSodium currents and calcium currents in trigeminal ganglion neurons were monitored using whole-cell patch-clamp recordings. Paired Student's t test was used as appropriate to evaluate the statistical significance of differences between two group means.

RESULTSBoth tetrodotoxin-resistant sodium currents and high-voltage activated calcium currents were blocked by flunarizine in a concentration-dependent manner with the concentration producing half-maximal current block values of 2.89 µmol/L and 2.73 µmol/L, respectively. The steady-state inactivation curves of tetrodotoxin-resistant sodium currents and high-voltage activated calcium currents were shifted towards more hyperpolarizing potentials after exposure to flunarizine. Furthermore, the actions of flunarizine in blocking tetrodotoxin-resistant sodium currents and high-voltage activated calcium currents were use-dependent, with effects enhanced at higher rates of channel activation.

CONCLUSIONBlockades of these currents might help explain the peripheral mechanism underlying the preventive effect of flunarizine on migraine attacks.

Animals ; Calcium ; metabolism ; Cells, Cultured ; Female ; Flunarizine ; pharmacology ; Male ; Mice ; Patch-Clamp Techniques ; Sensory Receptor Cells ; drug effects ; metabolism ; Sodium ; metabolism ; Trigeminal Ganglion ; cytology ; drug effects ; metabolism

OBJECTIVETo investigate the effects of Hg2+ on voltage-dependent calcium channels and intracellular free calcium in trigeminal ganglion neurons of rats and explore the toxicity mechanism of Hg2+ on these neurons.

METHODSWhole cell patch-clamp technique was used to determine ICa of voltage-dependent calcium channels in trigeminal ganglion neurons of rats. Intracellular free calcium was measured to explore [Ca2+]i dynamic changes from a single cell level by laser scanning confocal microscopy and fluorescence probe techniques.

RESULTS0.01, 0.10, 1.00 and 10.00 micromol/L Hg2+ could reduce voltage-dependent calcium channel currents ICa by (1.80+/-0.32)%, (23.04+/-9.46)%, (58.20+/-7.90)% and (82.00+/-5.77)% in trigeminal ganglion neurons. The inhibiting effects reached their maximum in 5 minutes and could not be reversed significantly during wash with Hg2+-free solution. Also, 0.01, 0.10 and 1.00 micromol/L Hg2+ increased intracellular free calcium concentrations by (2.50+/-0.83)%, (82.81+/-35.36)% and (222.70+/-62.48)% in trigeminal ganglion neurons. Pre-administrated trigeminal ganglion neurons with nifedipine for 10 minutes could decrease the effects and delay the effecting time.

CONCLUSIONThe inhibition of Hg2+ on the voltage-dependent calcium channel currents ICa depends on voltage-dependent calcium channels. And the increase of intracellular free calcium concentration in trigeminal ganglion neurons induced by Hg2+ is related to the release of intracellular stored calcium. However, the relationship between them needs further investigation.

Animals ; Calcium ; metabolism ; Calcium Channels ; drug effects ; metabolism ; physiology ; Cells, Cultured ; Female ; Male ; Mercury ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Trigeminal Ganglion ; cytology ; drug effects ; metabolism ; physiology

The characteristics of purinoceptors in the membrane of rat trigeminal ganglion (TG) neurons were studied by using whole- cell patch clamp technique. The results showed that most of neurons examined (78.9%, 142/180) were responsive to ATP in a concentration-dependent manner; the others (21.1%, 38/180) were ATP insensitive. Of the ATP-sensitive cells, the majority (95.1%, 135/142) responded to ATP with an inward current, a few (2.1%, 3/142) with an outward current, and the rest (2.8%, 4/142) with biphasic current. Small sized cells (<30 mum) responded to ATP with a rapid desensitizing inward current and were highly sensitive to vanilloid; the medium sized cells (30~40 mum) responded to ATP with slow desensitizing inward current and were not sensitive to vanilloid; while the majority of large sized cells (>40 mum) did not respond to ATP and vanilloid. The waveform of ATP-activated inward currents was related to the cell diameter. The I-V curves for both small and medium sized cells manifested obvious inward rectification. Furthermore, we studied the kinetic features of ATP-activated currents and the effects of P2 purinoceptor agonists and antagonists on I(ATP). The findings suggest that ATP receptor-ion channels are expressed differently among different types of rat TG neurons.
Adenosine Triphosphate ; metabolism ; Animals ; Animals, Newborn ; Neurons ; metabolism ; physiology ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; Receptors, Purinergic P2X ; physiology ; Trigeminal Ganglion ; cytology ; metabolism ; physiology

AIMTo observe redox modulation of ion channel in trigeminal ganglion neurons by oxidants and reducing agents.

METHODSThe effects of oxidants and reducing agents on maxi-conductance calcium-activated potassium channel in cultured rat trigeminal ganglion neurons by using whole-cell patch-clamp technique.

RESULTSMethionine-specific oxidant chloramine-T (Ch-T) 1 mmol/L slightly increased the current amplitude and this enhancement did not antagonized by DTT. In contrast, cysteine-specific reagent 5, 5'-dithio-bis(2-nitrobenzoic acid) (DTNB) 500 micromol/L significantly decreased current amplitude of BK(Ca) channels. The effect was reversed by the reducing agent 2 mmol/L 1, 4-dithio-DL-threitol (DTT).

CONCLUSIONReactive oxygen species were definitely involved in regulation of native neuronal function via redox modulation of BK(Ca) channels, which are suggested to play compensatory roles under oxidative stress-related conditions.

Animals ; Cells, Cultured ; Large-Conductance Calcium-Activated Potassium Channels ; physiology ; Male ; Neurons ; metabolism ; physiology ; Oxidation-Reduction ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; Trigeminal Ganglion ; cytology ; physiology

AIMTo explore the characteristic of ATP-activated current in trigeminal ganglion (TG) neurons of rat.

METHODSWhole-cell patch-clamp was performed.

RESULTS(1) The majority (92.1%) of TG neurons responded to ATP applied externally with inward currents. We recorded three distinct ATP-activated currents: fast, slow and intermediate, which were concentration-dependent. (2) In general, the fast ATP-activated currents were distributed mainly in small-diameter TG neurons, the slow ATP-activated currents were distributed mainly in large-diameter TG neurons, and the intermediate ATP-activated currents were distributed mainly in intermediate-diameter TG neurons. (3) The time course of rising phase from 10% to 90% of the three distinct ATP-activated currents were as follows: fast: (33.6 +/- 4.5) ms; intermediate: (62.2 +/- 9.9) ms; slow: (302.1 +/- 62.0) ms, and that of desensitizing phase were (399.4 +/- 58.2) ms (fast), and > 500 ms (slow) respectively. (4) From the current-voltage relationship curves, it can be seen that the reversal potential values of the three distinct ATP-activated currents were the same, all being 0-5mV. And they all were characterized by inward rectification. (5) The dose-response curve for fast ATP-activated current shifted downwards as compared with the intermediate ATP-activated current, and that for the slow ATP-activated current shifted upwards.

CONCLUSIONThe EC50s of the three curves tended to be identical. The results suggested that three kinds of distinct ATP-activated currents could be mediated by various subtypes of P2X receptors assembled by different subunits, and the subtypes existed in TG neurons of different diameters and transmit different information.

Animals ; Cells, Cultured ; Membrane Potentials ; Neurons ; physiology ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; Receptors, Purinergic P2 ; metabolism ; Trigeminal Ganglion ; physiology

OBJECTIVETo evaluate the effect of static magnetic field on the expression of SP-mRNA in TG in rats.

METHODS44 Wistar rats aged 6-7 weeks were put into static magnetic field and were sacrificed at 1 h, 2 h, 6 h, 12 h, 24 h, respectively. In situ hybridization method was used to evaluate the changes of SP-mRNA expression at different time point.

RESULTSMany neurons in TG were marked with SP probes in each group, the expression of SP-mRNA increased remarkably in static magnetic field group. In this group, the percentage of SP-mRNA positive neurons in TG increased greatly in 1 h, reached its peak in 2 h, from then on, decrease of the percentage started slowly but a moderate percentage was kept until 24 h, which was thought to be enough to maintain orthodontic tooth movement. The tendency of control group was almost the same with that of experimental group. The expression of SP-mRNA was higher in experimental group within 2 h but became lower after 2 h as compared with control group, this indicated that magnetic field reduced the SP-mRNA expression and exerted restoring effect on trauma. There were significant differences between experimental groups and control group at different time points (P < 0.01).

CONCLUSIONThe expression of SP-mRNA in TG in rats increased significantly in static magnetic field.

Animals ; Electromagnetic Fields ; Female ; RNA, Messenger ; biosynthesis ; genetics ; radiation effects ; Random Allocation ; Rats ; Rats, Wistar ; Substance P ; biosynthesis ; genetics ; radiation effects ; Trigeminal Ganglion ; metabolism ; radiation effects

OBJECTIVETo investigate changes of two sodium channels, PN(3) and NaN, during orofacial pain by occlusal trauma in rat.

METHODSExpressions of PN(3) mRNA and NaN mRNA in trigeminal ganglion were tested during various periods of persistent occlusal trauma with reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSIn groups, including control, occlusal trauma groups, PN(3) mRNA and NaN mRNA were all expressed in trigeminal ganglion neurons. In the control group, there were similar density values bilaterally. In the occlusal trauma group, the density values in gel electrophoresis of PN(3) mRNA and NaN mRNA on the intervention side were slightly greater than those on the control side.

CONCLUSIONSThe stimulation of occlusal trauma upregulates expressions of PN(3) mRNA and NaN mRNA, which suggests the signal occurring and conduction of chronic pain by occlusal trauma have the same molecular mechanism of sodium channel as inflammatory pain.

Animals ; Dental Occlusion, Traumatic ; physiopathology ; Facial Pain ; etiology ; Male ; NAV1.8 Voltage-Gated Sodium Channel ; RNA, Messenger ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Sodium Channels ; biosynthesis ; genetics ; Trigeminal Ganglion ; metabolism

OBJECTIVETo investigate the involvement of transient receptor potential vanilloid receptor 1 (TRPV1) in the facial inflammatory pain in relation to thermal hyperalgesia and cold pain sensation.

METHODSFacial inflammatory pain model was developed by subcutaneous injection of turpentine oil (TO) into rat facial area. Head withdrawal thermal latency (HWTL) and head withdrawal cold latency (HWCL) were measured once a day for 21 d after TO treatment using thermal and cold measurement apparatus. The immunohistochemical staining, cell-size frequency analysis and the survey of average optical density (OD) value were used to observe the changes of TRPV1 expression in the neurons of the trigeminal ganglion (TG), peripheral nerve fibers in the vibrissal pad, and central projection processes in the trigeminal sensory nuclei caudalis (Vc) on day 3, 5, 7, 14, and 21 after TO injection.

RESULTSHWTL and HWCL decreased significantly from day 1 to day 14 after TO injection with the lowest value on day 5 and day 3, respectively, and both recovered on day 21. The number of TRPV1-labeled neurons increased remarkably from day 1 to day 14 with a peak on day 7, and returned back to the normal level on day 21. In control rats, only small and medium-sized TG neurons were immunoreactive (IR) to TRPV1, and the TRPV1-IR terminals were abundant in both the vibrissal pad and the Vc. Within 2 weeks of inflammation, the expression of TRPV1 in small and medium-sized TG neurons increased obviously. Also the TRPV1 stained terminals and fibers appeared more frequent and denser in both the vibrissal pad skin and throughout laminae I and the outer zone of laminae II (IIo) of Vc.

CONCLUSIONFacial inflammatory pain could induce hyperalgesia to noxious heat and cold stimuli, and result in increase of the numbers of TRPV1 positive TG neurons and the peripheral and central terminals of TG. These results suggest that the phenotypic changes of TRPV1 expression in small and medium-sized TG neurons and terminals might play an important role in the development and maintenance of TO-induced inflammatory thermal hyperalgesia and cold pain sensation.

Animals ; Cold Temperature ; Facial Pain ; chemically induced ; metabolism ; physiopathology ; Hot Temperature ; Immunohistochemistry ; Male ; Neurons ; cytology ; metabolism ; Pain Threshold ; physiology ; Rats ; Rats, Sprague-Dawley ; Statistics, Nonparametric ; TRPV Cation Channels ; metabolism ; Thermosensing ; physiology ; Trigeminal Ganglion ; cytology ; metabolism ; Turpentine ; administration & dosage