Hideharu Ikeda

Capsaicin-sensitive Aδ Fibers in Cat Tooth Pulp

How close a correlation there is between the conduction velocity and receptive properties of pulpal nerve fibers is still unclear. Our specific aims were to confirm whether: (1) capsaicin affects not only polymodal C fibers but A8 fibers as well, and (2) Aδ polymodal nociceptors exist in the tooth pulp. A total of 139 functional single cat tooth pulp nerve fibers was isolated for analysis, of which 21 were Ap, 37 C, and 81 AS fibers. The A8 fibers were divided into two groups: One (n = 38) consisted of those fibers whose conduction velocities were more than 2.0 m/s both inside and outside the tooth pulp, and the other (n = 43) consisted of those fibers whose intrapulpal conduction velocities were less than 2.0 m/s, with extrapulpal conduction velocities greater than 2.0 m/s. We used 82 fibers to record the neural response following the topical application of capsaicin for 60 min at increasing concentrations (1 nM, 100 nM, 10 μM) through thin dentin. Six of 25 slow Aδ, 10/20 C, and no Ap (0/11) or fast AS (0/26) fibers responded to 1 nM or 100 nM of capsaicin. When the three concentrations of capsaicin solution were applied in turn, the electrical threshold and latency of Aβ and fast AS fibers did not change, whereas those of slow AS and C fibers gradually increased. In 0/11 Ap, 0/26 fast AS, 13/25 slow AS, and 18/20 C fibers, the conduction was blocked reversibly or irreversibly following the application of 10 uM of capsaicin. The amplitude of the late component of antidromic action potential of fast AS fibers decreased after the capsaicin application. No neural discharge could be recorded from 19 (3 Aβ, 5 fast AS, 6 slow AS, and 5C) fibers following the application of a single high concentration of capsaicin (10 μM). A single low concentration of capsaicin (100 nM) activated only some slow-conducting fibers (0/4 Ap, 0/4 FAS, 3/6 SAS, and 4/6 C). Response properties recorded from the remaining 18 fibers (3 Ap, 3 fast AS, 6 slow Aδ, and 6 C) were not changed following the application of the control vehicle. These results confirm that a low concentration of capsaicin has an excitatory effect on the response of slow pulpal AS as well as C fibers, and that a high concentration of capsaicin blocks the conduction of slow Aδ and C fibers as well as the terminals of fast AS fibers in the pulp.

Importance of clinical examination and diagnosis: A case of dens invaginatus

A case report is presented that shows a radiolucent lesion between the maxillary left lateral incisor and canine tooth, both of which were vital. The cause of the lesion was initially unclear. The patient was to be treated surgically, however, precise examination made it clear that the cause of the radiolucent lesion was due to the exposure of a second canal because of attrition of the lateral incisor crown. The final diagnosis of the tooth was dens invaginatus. This case highlights the importance of careful examination and correct diagnosis.

Subjective sensation and objective neural discharges recorded from clinically nonvital and intact teeth

The aim of this study was to compare subjective sensation with objective neural discharges recorded by microneurography. We examined human teeth that did not respond to pulp vitality testing, but that responded to cavity preparation for endodontic treatment (pathophysiological). Intact teeth and endodontically obturated teeth were used as controls. Pulpal blood flow in the clinical crown and histological examination were also used. Most teeth, both in normal and pathophysiological conditions, did not respond to all pulp vitality tests. Even when teeth in the pathophysiological group showed spike discharges evoked by pulp vitality tests or from spontaneous activity no sensation was elicited. These results confirmed the usefulness of microneurography for research on pulpal sensation and the significance of summation in the perception of sensation in chronically inflamed tooth pulp.

Sensory experiences in relation to pulpal nerve activation of human teeth in different age groups

There are no data on the correlation of intradental nerve activity and sensation from intact human teeth. We used microneurography to examine this relation and to determine whether it changes with age. Fifteen informed and healthy male volunteers were divided into three age groups: group A (18.6+/-1.82 years, mean+/-standard deviation (S.D.)), group B (38.4+/-2.70 years) and group C (64.0+/-4.06 years). Ratings of perceived pain intensity to thermal stimulation were obtained using a visual analogue scale (VAS). In addition, each subject chose one or two words from the short-form McGill Pain Questionnaire to describe perceived pain. A total of 90 single pulpal axons were studied with microneurography at the same time as the sensory experiences were recorded. Mean conduction velocities and variance estimates correlated closely with age. With advancing age, first, the percentage of teeth from which the subjects did not perceive any sensations to thermal stimulation increased, second, units responding to heat stimulus decreased, and third, latencies of sensation induced by thermal stimulation increased. In addition, a burst of afterdischarges following thermal stimulation and neural discharges evoked by thermal stimulation produced no sensation only in some of group B and C units. In contrast, no significant difference was found among three groups in VAS scores and words to describe the perceived pain to thermal stimulation. These results suggest that pulpal afferents were activated by the same mechanism(s); the hydrodynamic mechanism works immediately after thermal stimulation and is possibly followed by direct activation of some nerves, especially slow conducting fibres. In older tooth pulps, the decrease in the number of fast conducting afferents and mineral apposition of dentinal tubules impaired the nerve activation, especially by heat, as per the hydrodynamic mechanism. Spike discharges without sensation in older individuals were suggested to be due to insufficient spatial and temporal summation and may be involved with abnormal uncomfortable sensations.

Odontoblastic Syncytium through Electrical Coupling in the Human Dental Pulp

We have previously reported a dye-coupling network between odontoblasts (OBs). However, it is still unclear how the information detected by the odontoblasts is transmitted. The aim of this study was to characterize the odontoblastic syncytium electrophysiologically in the human dental pulp. Pulpal cells were freshly isolated from human premolars immediately after extraction. Under a light microscope, coupled or small clusters (3-20) of odontoblasts, each of which had a monopolar process (95-280 µm) and an oval cell body, were easily observed to be lined up in parallel. Cells were used for electrophysiological recording within 3 hrs in the dual patch-clamp configuration. Electrical couplings were found between odontoblasts (37/40 pairs). Voltage gating showed directional independence between pairs of odontoblasts. The time constant to a current decay increased with the number of clustered odontoblasts. Nine of 37 pairs isolated from young patients were electrically coupled, but could not be voltage-clamped. Transjunctional currents were blocked by octanol. These results suggest that odontoblasts form a syncytium that is directionally independent via symmetric gap junction channels in the odontoblastic layer. Young odontoblasts with a high electrical conductance to neighboring cells may be related to high potential of information transmission or calcification.

Facilitatory effect of AC-iontophoresis of lidocaine hydrochloride on the permeability of human enamel and dentine in extracted teeth

OBJECTIVES The objectives of the present study were to quantitatively evaluate chemical permeability through human enamel/dentine using conductometry and to clarify if alternating current (AC) iontophoresis facilitates such permeability. MATERIALS AND METHODS Electrical impedance of different concentrations of lidocaine hydrochloride was measured using a bipolar platinum impedance probe. A quadratic curve closely fitted to the response functions between conductance and lidocaine hydrochloride. For analysis of the passage of lidocaine hydrochloride through human enamel/dentine, eight premolars that were extracted for orthodontic treatment were sectioned at the cemento-enamel junction. The tooth crowns were held between two chambers with a double O-ring. The enamel-side chamber was filled with lidocaine hydrochloride, and the pulp-side chamber was filled with extrapure water. Two platinum plate electrodes were set at the end of each chamber to pass alternating current. A simulated interstitial pulp pressure was applied to the pulp-side chamber. The change in the concentration of lidocaine hydrochloride in the pulp-side chamber was measured every 2min using a platinum recording probe positioned at the centre of the pulp-side chamber. Passive entry without iontophoresis was used as a control. RESULTS The level of lidocaine hydrochloride that passed through enamel/dentine against the dentinal fluid flow increased with time. Electrical conductance (G, mho) correlated closely to the concentration (x, mmol/L) of lidocaine hydrochloride (G=2.16x(2)+0.0289x+0.000376, r(2)=0.999). CONCLUSIONS Lidocaine hydrochloride can pass through enamel/dentine. Conductometry showed that the level of lidocaine hydrochloride that passed through enamel/dentine was increased by AC iontophoresis.

The effect of pulpward pressure on the response to 50% lidocaine (lignocaine) applied to exposed dentine in cats

The responses of intradental nerves can be blocked by 50% lidocaine applied to exposed, etched dentine. The aim here was to analyse electrophysiologically the contribution of pulpward pressure to the anaesthetic effect of lidocaine applied to exposed dentine. In 17 adult cats with intact lower canines, approximately 1mm of the coronal tip of the canine was removed and the exposed dentine acid-etched. Pulpward pressure was applied to the exposed dentine through a chamber fixed to the tip of the tooth. Cervical and coronal electrodes of silver wire and Ag/AgCl mixture were placed, the cervical 2mm from the gingival margin of the canine and the coronal into the coronal pulp chamber. The inferior alveolar nerve was surgically exposed and single pulpal nerve fibres responding to electrical stimulation of the canine were identified. The chamber was filled with 50% lidocaine solution, and 0 (atmospheric pressure), 1.3, 5.0 or 10.0 kPa pressure was applied to the chamber for 20 min. Orthodromic and antidromic action potentials were compared before and after the lidocaine application. When lidocaine was applied at 0, 1.3 or 5.0 kPa (n=5 each) for 20 min, all the orthodromic responses to cathodal electrical stimulation with the coronal electrode were blocked. Almost all orthodromic responses (12/15) remained unblocked when the cathodal electrical stimulation was applied with the cervical electrode. Applying 10.0 kPa pressure to the lidocaine solution blocked all the pulpal nerve responses (n=5) to both stimulation modes. The amplitude of the antidromic compound action potential was significantly decreased when the applied pressure was increased. These results suggest that increased pulpward pressure may effectively force anaesthetic solution more rapidly through the dentinal tubules into the pulp.

THREE GROUPS OF AFFERENT PULPAL FELINE NERVE FIBRES SHOW DIFFERENT ELECTROPHYSIOLOGICAL RESPONSE PROPERTIES

Responses were recorded, after the application of four types of stimuli (slow or rapid elevation of temperature, hydrostatic negative pressure through thin dentine, and bradykinin directly applied to exposed pulp), from functional single fibres innervating the cat lower canine tooth pulp, dissected from the inferior alveolar nerve. A total of 278 single pulpal fibres were isolated. A fibres (n = 220) were divided into two groups: one (FA; fast A fibre, n = 160) consisting of those whose conduction velocities (CVs) were more than 2 m/s both inside and outside the tooth pulp, and the other (SA delta; slow A delta fibre, n = 60) consisting of those whose intrapulpal CVs were less than 2 m/s and extrapulpal CVs greater than 2 m/s. Fifty eight C fibres (C) were also found. None of FA, 40% of SA delta and 52% of C responded to continuous heat. None of C, 47% of FA and 45% of SA delta responded to rapid elevation of temperature. None of C, 20% of FA and 20% of SA delta responded to hydrostatic pressure. None of FA, 83% of SA delta and all of C responded to bradykinin. It was found that 21 of 60 SA delta responded to both types of stimuli that reportedly activate only A (rapid heat and hydrostatic negative pressure) or C (continuous slow heat and bradykinin) nerve fibres and that 29 SA delta responded to slow heating and/or bradykinin, similar to C fibres.

Transient receptor potential melastatin (TRPM) 8 is expressed in freshly isolated native human odontoblasts

OBJECTIVE Cold-sensitive ion channels, such as transient receptor potential melastatin (TRPM) 8 and transient receptor potential ankyrin (TRPA) 1, may play a crucial role in the nociceptive function of odontoblasts, whereas expression of these TRP channels in human native odontoblasts remains to be elucidated. This study aimed to analyze the expression of TRPM8 and TRPA1 in freshly isolated native human odontoblasts. DESIGN Odontoblasts were isolated from freshly extracted healthy human teeth (n=4); after removing the inner pulp tissues from the pulp chambers, odontoblasts remaining on the dentin surface were washed out with phosphate buffered saline and collected. Reverse transcription-polymerase chain reaction was employed to compare the expression levels of TRPM8, TRPA1, and dentin matrix acidic phosphoprotein 1 (DMP1) mRNAs between the isolated odontoblasts and the inner pulp tissues. The isolated cells were subjected to immunolocalization of TRPM8 and nestin. Paraformaldehyde-fixed, EDTA-demineralized frozen sections obtained from freshly extracted healthy human teeth (n=4) were also analyzed immunohistochemically using anti-nestin, TRPM8, and TRPA1 antibodies. RESULTS Expression levels of TRPM8 and DMP1 in the isolated odontoblasts were significantly higher than those in the inner pulp tissues (p<0.05). Expression of TRPM8 and nestin was observed in the odontoblastic layer of the dental pulp tissue and isolated odontoblasts, while expression of TRPA1 was not detected. CONCLUSIONS TRPM8, but not TRPA1, was detected in freshly isolated native human odontoblasts at the protein and mRNA levels, suggesting that odontoblasts play an important role in detecting external cold stimulation via TRPM8 in healthy condition.

Determination of the functional space for fluid movement in the rat dentinal tubules using fluorescent microsphere.

OBJECTIVES The size of the functional space available for hydrodynamic fluid movement between cellular components and the walls of dentinal tubules has not yet been investigated. We attempted to measure the space using small diameter fluorescent microspheres. METHODS The coronal enamel of 144 rat molars was removed to expose the dentine, which was acid-etched. Fluorescent microspheres of different diameters (0.02-4.0μm) were applied to the exposed dentine for 60min before the rat jaws were cut into cryostat sections. The distribution and fluorescent intensities of the fluorescent microspheres were examined with confocal laser scanning microscope and analyzed using image analysis software. RESULTS Microspheres with a diameter of 2.0-4.0μm were detected only on the surface of the cavities. A small number of microspheres with a diameter of 1.0μm accumulated primarily in the outer third of the dentine. Microspheres with a diameter of 0.2-0.5μm were found in the outer and middle thirds of the dentine. Microspheres with a diameter of 0.02-0.1μm accumulated in the middle and occasionally inner thirds of the dentine. Some of the microspheres measuring 0.02-0.04μm in diameter reached the dental pulp. CONCLUSIONS The dentinal tubules in the inner third of the rat coronal dentine may have a space less than 0.1μm through which dentinal fluid can move, despite outward tapering of the dentinal tubules. Retrograde tapering may increase the pressure in the inner third of the dentine layer, and this elevated pressure may contribute to mechanical deformation of the content in the dentinal tubules.