Immunohistochemical localization of SNARE core proteins in intrapulpal and intradentinal nerve fibers of rat molar teeth.

OBJECTIVE: The present study was designed to elucidate whether three soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) core proteins, syntaxin-1, synaptosomal-associated protein of 25kDa (SNAP-25), and vesicle-associated membrane protein-2 (VAMP-2), are present in the dental pulp of the rat molar at both the light and electron microscopic levels. DESIGN: Immunohistochemistry for protein gene product 9.5 (PGP 9.5), a pan-neuronal marker, syntaxin-1, SNAP-25, and VAMP-2 was performed on decalcified rat molars for light and electron microscopic analyses. Double-immunolabeling of PGP 9.5 and the SNARE core proteins, as well as combinations of the SNARE core proteins, was also carried out. RESULTS: PGP 9.5-immunoreactive nerve fibers ran toward the coronal region, ramified at the subodontoblast layer, and formed the subodontoblastic nerve plexus. Most nerve fibers penetrated the predentin and dentin along the dentinal tubules. Most, if not all, nerve fibers displayed immunoreactivity for syntaxin-1, SNAP-25, and VAMP-2. Immunoelectron microscopic analyses confirmed the presence of immunoreactivity for the SNARE core proteins within the intradental axonal elements. CONCLUSIONS: The present findings suggest that, since SNARE core proteins participate in the docking and exocytosis of synaptic vesicles in the central nervous system, they may contribute to vesicle exocytosis from the dental nerve fibers even though there are no apparent synapses.

Neurotoxicity of various root canal sealers on rat sciatic nerve: an electrophysiologic and histopathologic study.

OBJECTIVES: The aim of this study was to compare the neurotoxicity of various root canal sealers on rat sciatic nerve by electrophysiologic and histopathologic analyses. MATERIALS AND METHODS: A total of 40 male rats were randomly divided into five groups: Control, AH Plus, GuttaFlow, Sealapex and Smartpastebio. Sciatic nerves of the rats were uncovered using the surgical procedures, and the prepared sealers were then applied on nerves with a polyethylene tube vehicle for 15 days. Nerve potentials were recorded at initial exposure, 5, 30 and 120 min (early phase), and 15 days (late phase) by an electrophysiologic analysis system for all groups. The obtained measurements were then used to calculate the nerve conduction velocities (NCV). Subsequently, all rats were sacrificed, and their sciatic nerves were removed for histopathologic analysis. Statistical analysis was performed using the Kruskal-Wallis and Mann-Whitney U tests for intergroup variables and the Friedman and Wilcoxon test for intragroup variables. Statistical significance was set at P < 0.05. RESULTS: There was no significant difference between early and late phase results in the control group. This group showed little or no lasting damage to nerve tissue. All sealers decreased the NCV in the early phase time periods, but this decrease was only statistically significant in the AH Plus group at 120-min time period (P < 0.0125). During the late phase, the AH Plus and GuttaFlow groups almost reached initial NCV values, and it was lower than the initial values in the Sealapex and Smartpastebio groups. However, this decrease was not statistically significant. When intergroup comparisons were performed, statistically significant differences occurred at 30 min in the Sealapex group and 120 min in the AH Plus group compared with the control group (P < 0.0125). All sealers induced neurotoxicity as a result of degenerative and inflammatory responses of nerve tissue in histologic analysis. Histologic analysis revealed Sealapex and GuttaFlow to be the most and least neurotoxic, respectively. CONCLUSIONS: All tested root canal sealers exhibited a variable degree of neurotoxicity depending on their chemical compositions. CLINICAL RELEVANCE: Apical extrusion of endodontic filling materials may cause undesired consequences, such as inflammation and severe neurotoxic damage; therefore, extrusion factor plays an important role during the root canal treatment.

Effect of nitrous oxide on the efficacy of the inferior alveolar nerve block in patients with symptomatic irreversible pulpitis.

INTRODUCTION: The inferior alveolar nerve (IAN) block does not always result in successful pulpal anesthesia. Anesthetic success rates might be affected by increased anxiety. Nitrous oxide has been shown to have both anxiolytic and analgesic properties. Therefore, the purpose of this prospective, randomized, double-blind, placebo-controlled study was to determine the effect of nitrous oxide on the anesthetic success of the IAN block in patients experiencing symptomatic irreversible pulpitis. METHODS: One hundred emergency patients diagnosed with symptomatic irreversible pulpitis of a mandibular posterior tooth were enrolled in this study. Each patient was randomly assigned to receive an inhalation regimen of nitrous oxide/oxygen mix or room air/oxygen mix (placebo) 5 minutes before the administration of the IAN block. Endodontic access was begun 15 minutes after completion of the IAN block, and all patients had profound lip numbness. Success was defined as no or mild pain (visual analog scale recordings) on access or instrumentation. RESULTS: The success rate for the IAN block was 50% for the nitrous oxide group and 28% for the placebo group. There was a statistically significant difference between the 2 groups (P = .024). CONCLUSIONS: For mandibular teeth diagnosed with symptomatic irreversible pulpitis, administration of 30%-50% nitrous oxide resulted in a statistically significant increase in the success of the IAN block compared with room air/oxygen.

Effect of tooth pulp and periaqueductal central gray stimulation on the expression of genes encoding the selected neuropeptides and opioid receptors in the mesencephalon, hypothalamus and thalamus in rats.

Nociceptive stimulation has been considered to affect the expression of genes encoding endogenous neuropeptides and their receptors. The effect of electric stimulation of the tooth pulp and/or periaqueductal gray (PAG) in rats on mRNA levels of the selected neuropeptides and opioid receptors (ORs) was investigated in comparison with control group, without stimulation. The levels of mRNA for the selected neuropeptides: galanin (GAL), vasopressin (AVP), oxytocin (OT), substance P (SP), somatostatin (SOM), vasoactive intestinal peptide (VIP), endomorphin-2 (EM-2), and opioid receptors: MOR, DOR and KOR in mesencephalic, hypothalamic and thalamic tissues were determined by real-time PCR. It was demonstrated that in the control group expression of the tested neuropeptides was at a very low level in the mesencephalon and thalamus, but at the higher level in the hypothalamus. The highest expression of ORs was observed in the mesencephalon. Nociceptive tooth pulp stimulation had the strongest effect in the hypothalamus, elevating mRNA levels of all tested neuropeptides except SOM. Electric stimulation of PAG either did not change or down-regulated mRNA levels of the neuropeptides in the cerebral structures. Simultaneous stimulation of PAG and tooth pulp either did not affect mRNA levels of the investigated neuropeptides or caused their slight decrease versus tooth pulp stimulation. The noxious stimulation of tooth pulp increased also the levels of OR mRNAs, while stimulation of PAG had the opposite effect. The above results demonstrated that tooth pulp stimulation significantly up-regulated the mRNA levels for a number of neuropeptides and all three types of ORs in the rat brain, which would result in more potent antinociception. In contrast, PAG stimulation down-regulated the mRNA levels of several neuropeptides and ORs in the cerebral tissues, which would cause decreased synthesis of ORs. The obtained results represent a new insight into the mechanism of orofacial pain.

Pulpitis increases the proportion of atypical nodes of Ranvier in human dental pulp axons without a change in Nav1.6 sodium channel expression.

Studies show a change in sodium channel (NaCh) expression after inflammatory lesions, and this change is implicated in the generation of pain states. We are using the extracted human tooth to study NaCh expression and here examine the expression of the major NaCh isoform located at nodes of Ranvier, Na(v)1.6, in normal and painful samples. Pulpal sections were double-labeled with human-specific Na(v)1.6 antibody and caspr antibody (paranodal protein to identify nodes). Confocal microscopy was used to obtain a z-series of optically-sectioned images of axon bundles surrounded by inflammatory cells in painful samples and of similar regions within the coronal pulp of normal samples. Nodes contained within these images were classified as typical or atypical as based on caspr staining relationships, and NIH ImageJ software was used to quantify the size and immunofluorescence staining intensity of Na(v)1.6 accumulations at these nodal sites. Results show no significant difference in the size or immunofluorescence staining intensity of Na(v)1.6 nodal accumulations located at either typical or atypical nodal sites (heminodes and split nodes) within axons in normal samples when compared to painful samples (n=9/each group). In contrast, there was a highly significant decrease in the proportion of typical nodal sites and an increase in atypical nodal sites in painful samples when compared to normal samples. The unchanged expression of Na(v)1.6 contrasts to our previous finding that showed an increased expression of Na(v)1.7 at both typical and atypical nodal sites within painful samples. Together, these findings suggest there is not a simple replacement of one isoform with another, but rather an increased co-expression of multiple isoforms at both intact and remodeling/demyelinating (atypical) nodal sites within the painful dental pulp. The resultant heterogeneous population of isoforms may produce unique axonal excitability properties that could contribute to spontaneous pain sensations that are common in toothache.

Theophylline attenuates hippocampal blood flow responses induced by tooth pulp stimulation in rats.

In this study, we performed tests to determine whether tooth pulp stimulation (TPS) increases hippocampal blood flow (HBF), and if so, to investigate whether the increase in HBF is mediated via the activation of adenosine receptors. We measured HBF in urethane-anesthetized rats using laser Doppler flowmetry (LDF) and examined the effect of theophylline, a nonselective adenosine receptor antagonist, on TPS-induced HBF responses. TPS increased HBF, and its response was significantly attenuated by the intraperitoneal administration of theophylline (20 mg/kg). These results suggest that the HBF response induced by TPS may be, at least in part, produced through adenosine receptors.

Involvement of NOS/NO in the development of chronic dental inflammatory pain in rats.

Nitric oxide (NO) is believed to be an important messenger molecule in nociceptive transmission. To assess the possible roles of NO in trigeminal sensory system, we examined the distribution and density of histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), a marker for nitric oxide synthase (NOS), and immunohistochemical staining for c-Fos, a neuronal activity marker, in the trigeminal ganglion (TG) and trigeminal nucleus caudalis (Vc) following pulp exposure (PX) injured rats. The neurons innervating injured tooth in TG were labeled by the retrograde transport of fluoro-gold (FG). Teeth were processed for H&E staining. We found that NADPH-d activity increased significantly in the TG and Vc following PX pretreatment (7-28 days, especially in 21-28 days). Such changes were closely corresponding to the pattern of c-Fos detected by immunocytochemistry. The results demonstrate that PX-induced chronic pulpal inflammation results in significant alterations in the TG cells and in the Vc, and such changes may underlie the observed NADPH-d activity. It suggests that NOS/NO may play an active role in both peripheral and central processing of nociceptive information following chronic tooth inflammation.

Sympathetic nerve fibers sprout into rat odontoblast layer, but not into dentinal tubules, in response to cavity preparation.

This study was designed to determine if sympathetic nerve fibers exist in dentinal tubules in rat normal dental pulp, and if they sprout into the dentinal tubules in response to artificial cavity preparation in dentin. Sympathetic nerve fibers in rat molar dental pulp were labeled using an anterograde axonal transport technique involving injection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the superior cervical ganglion (SCG). They were then observed using light and electron microscopes. In normal dental pulp (control), scattered WGA-HRP reaction products were observed in unmyelinated nerve endings in the odontoblast layer and subodontoblastic region. In injured pulp 3 weeks after cavity preparation, reaction products were about 1.8-times more plentiful in the above areas (versus control pulp). However, no labeled nerve fibers were observed in the dentinal tubules in either control or injured dental pulp. These results indicate that although sympathetic nerve fibers do indeed sprout in rat dental pulp in response to cavity preparation, they do not penetrate into the dentinal tubules in which postganglionic nerve endings derived from the SCG were not originally present.

Articaine for supplemental intraosseous anesthesia in patients with irreversible pulpitis.

The purpose of this study was to determine the anesthetic efficacy and heart rate effect of 4% articaine with 1:100,000 epinephrine for supplemental intraosseous injection in mandibular posterior teeth diagnosed with irreversible pulpitis. Thirty-seven emergency patients, diagnosed with irreversible pulpitis of a mandibular posterior tooth, received an inferior alveolar nerve block and had moderate-to-severe pain upon endodontic access. The Stabident system was used to administer 1.8 ml of 4% articaine with 1:100,000 epinephrine. Success of the intraosseous injection was defined as none or mild pain upon endodontic access or initial instrumentation. The results demonstrated that anesthetic success was obtained in 86% (32 of 37) of the patients. Maximum mean heart rate was increased 32 beats/minute during the intraosseous injection. We can conclude that when the inferior alveolar nerve block fails to provide profound pulpal anesthesia, the intraosseous injection of 4% articaine with 1:100,000 epinephrine would be successful 86% of the time in achieving pulpal anesthesia in mandibular posterior teeth of patients presenting with irreversible pulpitis.

Comparison of Fos expression within the ferret's spinal trigeminal nuclear complex evoked by electrical or noxious-thermal pulpal stimulation.

UNLABELLED: Fos-like immunoreactivity (Fos-LI) was used as a marker of trigeminal neurons that responded to tooth pulp stimulation. The activation of intradental afferents was produced by electrical stimulation of the ferret's intact canine tooth, whereas natural stimuli that activate predominantly Adelta (5 mol/L CaCl2 applied to dentin) or C fibers (slow heating of the intact tooth) were used to stimulate the 2 populations of afferents selectively. Electrical stimulation evoked Fos-LI in ipsilateral dorsomedial of the trigeminal subnucleus caudalis (Vc), dorsomedial and ventrolateral of the transition zone between subnucleus interpolaris and caudalis (Vi/Vc), and the the paratrigeminal nucleus (Pa5). Osmotic stimulation evoked Fos-LI in the ipsilateral dorsomedial Vc and Vi/Vc. The spatial distribution of Fos labeling after heat stimulation was dependent on the duration and location of the stimulus application. Repeated heating of the maxillary canine for 30 minutes evoked labeling bilaterally in ventrolateral Vi/Vc. Stimulation of the maxillary and mandibular canines with heat pulses for 1 hour produced labeling in the ipsilateral dorsomedial Vc, dorsomedial Vi/Vc, and the Pa5. None of the stimulating procedures did evoke Fos expression in regions rostral to Vi/Vc. Regardless of the pulpal stimulation procedures, a similar number of Fos-positive neurons was found in the nucleus of solitary tract and the ventrolateral medulla. Although Fos expression does not reveal all neurons that respond to noxious pulpal stimulation, it marks many neuronal regions that contain neurons that respond to pulpal stimulation and injury. Our results suggest that a population of neurons in Vc and Vi/Vc contribute to painful sensations originating from the dentition. PERSPECTIVE: We demonstrated that the trigeminal nucleus caudalis and the transitional zone between trigeminal interpolaris and caudalis mediate painful sensation in the dental pulp. Both trigeminal regions might be therapeutic targets for dental pain in the future.

Role of capsaicin-sensitive primary afferent inputs from the masseter muscle in the C1 spinal neurons responding to tooth-pulp stimulation in rats.

The aim of the present study was to demonstrate the convergence of inputs from masseter muscle (MM) and tooth pulp (TP) onto C1 spinal neurons and to determine whether the afferent fibers express the functional vanilloid receptor (VR1). Extracellular single-unit recordings were made from 61 C1 units responding to TP electrical stimulation with a constant temporal relationship to a digastric electromyogram signal in pentobarbital anesthetized rats. Eighty-four percent of C1 neurons responding to TP stimulation also responded to the ipsilateral MM stimulation. Of these neurons, 61% were considered to be afferent inputs from Adelta-fibers and the remaining units (39%) were C-fibers, based on calculation of the nerve conduction velocity. Intramuscular injection of capsaicin (0.05 and 0.1%) produced a reduction in a MM-induced C1 neuronal activity in a dose-dependent manner and this effect was antagonized by pretreatment with an antagonist of VR1, capsazepine. Some of these units were also excited by noxious heat stimulation (> 43 degrees C). The trigeminal root ganglion (TRG) neurons that innervated the MM were retrogradely labeled with Fluorogold (FG) and the small-diameter FG-labeled TRG neurons expressed the immunoreactivity for VR1. After intramuscular mustard oil injection (noxious chemical stimulation), the C1 neuronal activity induced by both touch and pinch stimuli was enhanced and their receptive field sizes were significantly expanded. These changes were reversed within 15-20 min. These results suggest that there may be the convergence of noxious afferents inputs from the MM and TP afferents on the same C1 neurons in rats, and that the afferent fibers expressing the functional VR1 may contribute to the hyperalgesia and/or referred pain associated with temporomandibular joint disorder.

The co-expression of P2X3 receptor with VR1 and VRL-1 in the rat trigeminal ganglion.

The co-expression of P2X3 receptor with the vanilloid receptor subtype I (VR1) and vanilloid receptor 1-like receptor (VRL-1) was examined in the rat trigeminal ganglion (TG) by a double immunofluorescence method. P2X3 receptor-immunoreactive (ir) neurons were predominantly small to medium-sized (range=93.8-1844.4 microm(2), mean+/-S.D.=503.8+/-286.5 microm(2)); 35% and 9% of P2X3 receptor-ir TG neurons were immunoreactive for VR1 and VRL-1, respectively. Small and medium-sized P2X3 receptor-ir neurons contained VR1-immunoreactivity (ir), whereas medium-sized and large P2X3 receptor-ir neurons showed VRL-1-ir. The retrograde tracing and immunohistochemical methods revealed that 30% of the TG neurons retrogradely labeled from the facial skin and tooth pulp exhibited P2X3 receptor-ir. The co-expression of P2X3 receptor and VR1 was detected in 16% of cutaneous TG neurons and 6% of tooth pulp neurons. On the other hand, the co-expression of P2X3 receptor and VRL-1 was common in tooth pulp neurons (23%) and rare in cutaneous TG neurons (8%). In the tooth pulp, 95% of P2X3 receptor-ir TG neurons contained VRL-1-ir. The present study indicates that P2X3 receptor-ir TG neurons, which co-express VR-ir, are abundant in the facial skin. The tooth pulp is probably innervated by TG neurons, which contain both P2X3-and VRL-1-ir.

The co-expression of ASIC3 with calcitonin gene-related peptide and parvalbumin in the rat trigeminal ganglion.

The co-expression of ASIC3 with calcitonin gene-related peptide (CGRP) or parvalbumin (PV) was examined in the trigeminal ganglion (TG) by a double immunofluorescence method. ASIC3-immunoreactivity (IR) was detected in 23% of TG neurons. These neurons were of various sizes (range= 43-1768 microm(2), mean+/-S.D.=651+/-356 microm(2)); 26% and 14% of ASIC3-immunoreactive (IR) neurons co-expressed CGRP- and PV-immunoreactivity (IR), respectively; 33% and 13% of the TG neurons retrogradely labeled from the tooth pulp and facial skin, respectively, exhibited ASIC3-IR; 36% of CGRP-IR TG neurons which innervate these tissues co-expressed ASIC3-IR. Only 4% of ASIC3-IR cutaneous TG neurons showed PV-IR, while 25% of ASIC3-IR tooth pulp neurons were also immunoreactive for PV. The present study suggests that ASIC3-IR TG neurons supply the tooth pulp and facial skin with unmyelinated or finely myelinated axons. ASIC3-IR neurons which have large myelinated axons may be common in the tooth pulp but not the facial skin. The axonal morphology of ASIC3-IR TG neurons may depend on the variety of their receptive fields.

Nociceptive behaviour induced by dental application of irritants to rat incisors: a new model for tooth inflammatory pain.

Animal models simulating acute human pulpitis are still lacking. The rat incisors present a particular situation where most of their innervation is considered to be unmyelinated and concentrated mainly in the tooth pulp. This study reports on a new model for dental pain induced by inflammatory agents applied to the tooth pulps of incisors. In different groups of rats, artificial crowns were fixed on the lower incisors, after cutting 1-2mm of their distal extremities. A volume of 7-10 microl of solutions of saline, capsaicin (1-10mg/ml) or formalin (2.5% or 5%) was injected in the crown cavity, and the nociceptive behaviour was quantitated following a devised scoring method of four scales. Intradental application of capsaicin produced nociceptive scores in the form of one plateau for 1-2h depending on the concentration used. Similar results were obtained with intradental application of formalin 2.5%. The one plateau of nociceptive scores obtained with formalin contrasts with the biphasic aspect of nociceptive behaviour described with the intradermal formalin test. This discrepancy could be attributed to a difference in the types of afferent fibres involved in each situation. Pretreatment with morphine (2 mg/kg) attenuated, in a naloxone-reversible manner, the nociceptive behaviour observed following intradental application of capsaicin. Pretreatment with meloxicam (a cyclo-oxygenase-2 inhibitor) exerted a less pronounced attenuation of the nociceptive scores when compared with morphine. These results provide evidence for the validity of the described model for the simulation of tooth pulp inflammatory pain in awake animals.

Effects of pulsed Nd:YAG laser radiation on action potential conduction in nerve fibres inside teeth in vitro.

OBJECTIVE: This study aimed to simulate the effects of lasing dentine on pulpal nerve function. METHODS: Rat spinal nerve roots were threaded through the prepared pulp canal of a 10 mm long tooth root segment which was mounted in a perspex bath. The protruding ends of the nerve were placed on platinum wire electrodes used to elicit and to record compound nerve action potentials (CAPs). Laser energy (average power = 0.3-3.0 W) was applied to the surface of the root segment using a pulsed Nd:YAG dental laser (dLase 300). RESULTS: With the laser probe tip placed in static contact with the tooth surface, the nerve CAP was irreversibly abolished within 60 s of lasing at 1.0-3.0 W power. When the laser tip was moved to and fro over the root surface in a scanning mode, similar levels of radiation produced less marked effects. In the latter mode, CAP attenuation increased with increasing power and duration of lasing. After 60 s lasing at 0.3 W, the CAP size was 95% (+/- 5, S.D.) of the prelasing controls value; with 2.0 W the CAP was reduced to 54% (+/- 33). The CAP recovered to 90% of control levels after lasing at powers up to 1.5 W, but reached only 72% of control values after lasing at 2.0 W power. CONCLUSIONS: Laser radiation applied to dentine caused a dose-dependent block of action potential conduction in nerve fibres in the underlying pulp chamber.

Reinnervation of developing rat molars.

The ability of regenerating inferior alveolar nerve (IAN) fibres to reinnervate dentine of developing rat first molar teeth was investigated. At intervals of 5, 15, 30 and 50 days after intramandibular transection of the IAN at the age of 20 days, the percentage of innervated dentinal tubules was estimated and compared with results from a series of control specimens. In addition, the myelinated axon populations of the root canal pulps were examined by light microscopy. Degeneration of almost all pulpal myelinated axons and dentinal unmyelinated axons occurred within 5 days of surgery. By 15 days after transection there was evidence of some pulpal reinnervation by myelinated axons but less than 2% of dentinal tubules showed reinnervation (control, 31.8%). At 30 days after surgery the figure for dentinal reinnervation was approximately 17.7% (control, 44.9%), and by 50 days after transection (70 days of age) mean innervation was about 70% of the level observed in control 70 days teeth, though the difference between control and experimental specimens was not significant at the 5% level of probability. The results indicate that reinnervation of dentine does occur in developing teeth after nerve transection. It is argued that the results suggest a faster and probably more complete reinnervation in young animals; and that reinnervation may be attributable more to an active than to a passive mechanism, and this may also apply to dentinal innervation during development.

Projection of tooth pulp afferents to the brainstem and to the cortex in the cat.

The projection of tooth pulp afferents in the spinal trigeminal nucleus (N.V.sp.) and the effect of dorsolateral medullotomy on cortical potentials evoked by electrical stimulation of Adelta tooth pulp nerve fibres were studied in cats. It was confirmed that antidromic responses were recorded in the tooth pulp nerve to stimulation of the ipsilateral but not the contralateral N.V.sp.[11]. Dorsolateral medullotomy at the level of the obex did not alter the cortical potentials induced by single pulses applied to the tooth pulp. It is concluded that Adelta tooth pulp afferents project exclusively to the ipsilateral trigeminal nucleus and that the impulse transmission to the cortex persists after transection of the pars caudalis of the N.V.sp.