Mitsuhiro Sunakawa

Responsiveness changes of tooth pulp-driven neurons in thalamic ventral posteromedial and mediodorsal nuclei following experimental pulpitis and naloxone administration in rats

Abstract Aims: This study aimed to elucidate modifications of tooth pulp-driven neuron (TPDN) activities in thalamic ventral posteromedial (VPM) (n = 11) and mediodorsal (MD) (n = 13) nuclei following chemical conditioning stimulation with the inflammatory irritant mustard oil (allyl-isothiocyanate, MO) applied to pulp in rats. An additional aim was to examine the participation of the endogenous opioid peptiderelated central mechanism in this modification. Methods: Single unit activities of TPDNs were recorded extracellularly from the two thalamic nuclei, and changes in their responsiveness were tested during MO and mineral oil (Min) conditioning stimulation to the same pulp (VPM: n=8 ; MD: n=7). Results TPDNs in the VPM and MD showed significant increases in pulp-evoked responses following the MO conditioning stimulation (p U -test). Systemic administration of the opiate antagonist naloxone with MO conditioning stimulation produced different patterns of significant increases in VPM and MD TPDN responsiveness in respect to the spike numbers evoked. All TPDNs had cutaneous mechanoreceptive fields (CMRFs), and MO conditioning significantly lowered the mechanical threshold of responses to touch and pressure applied to the CMRF (p t -test). Conclusion: These results demonstrated that MO conditioning stimulation of pulp affects the responsiveness and cutaneous mechanical threshold of thalamic TPDNs suggesting that responsiveness of neurons in the central nervous system may be sensitized and modified by pulpal inflammation. Different types of modifications in neuronal responsiveness may be closely correlated with different mechanisms related to pain in tooth pulp.

Neuron-immune Interactions in the Sensitized Thalamus Induced by Mustard Oil Application to Rat Molar Pulp

We have reported that mustard oil application to the rat dental pulp induces neuronal activation in the thalamus. To address the mechanisms involved in the thalamic changes, we performed neuronal responsiveness recording, immunohistochemistry, and molecular biological analysis. After mustard oil application, neuronal responsiveness was increased in the mediodorsal nucleus. When MK801 (an N-methyl-D-aspartate receptor antagonist) was applied to the mediodorsal nucleus, the enhanced responsiveness was decreased. N-methyl-D-aspartate receptor 2D, glial fibrillary acidic protein, and antigen-presenting cell-related gene mRNAs in the contralateral thalamus were up-regulated at 10 minutes after mustard oil application, but were down-regulated within 10 minutes after the antagonist application. OX6-expressing microglia and glial fibrillary acidic protein-expressing astrocytes did not increase until 60 minutes after mustard oil application. These results suggested that the thalamic neurons play some roles in regulating the glial cell activation in the mediodorsal nucleus via N-methyl-D-aspartate receptor 2D during pulp inflammation-induced central sensitization.

Increased blood flow and nerve firing in the cat canine tooth in response to stimulation of the second premolar pulp

Mustard oil or mechanical stimulation was applied to maxillary second premolar tooth pulps and pulpal blood flow and or intradental nerve activity in the ipsilateral canine tooth were recorded in the cat. Mustard oil application to the second premolar pulp significantly increased blood flow in the canine tooth pulp to 162.0+/-65.8% (n = 16) of the prestimulation flow compared to control data obtained with application of mineral oil (107.0+/-5.1%, n = 6) (Mann-Whitney U-test, p = 0.0009). Sectioning of the infraorbital nerve and its branches on the experimental side (n = 4) did not affect this increase in pulpal blood flow. The paraperiosteal injection of 2% lidocaine (1.0 ml) without vasoconstrictor significantly inhibited the increase in canine pulpal blood flow induced by mustard oil application to the second premolar pulp (109.8+/-6.8% of the prestimulation level, n = 7) (Mann-Whitney U-test, p = 0.0013). Sporadic firing or sometimes bursts of action potentials in the canine pulp nerves were recorded during and/or after the mustard oil application to the second premolar pulp in three of 16 cases. Four single pulp nerve units firing in synchrony with the mechanical stimulation of the second premolar pulp were recorded in two of eight canines, which substantiated the existence of branched afferents innervating both teeth. These findings suggest that stimulation of the second premolar pulp may induce axon reflex-related vasodilation and intradental nerve firing in the canine pulp via branched afferent fibres innervating both the second premolar and canine teeth.

Artificial Dental Pulp Exposure Injury Up-regulates Antigen-Presenting Cell–related Molecules in Rat Central Nervous System

INTRODUCTION Bacterial infection and resulting inflammation of the dental pulp might not only trigger neuroimmune interactions in this tissue but also sensitize the central nervous system (CNS) such as the thalamus via nociceptive neurons. Thus, immunopathologic changes in the rat thalamus that take place after pulp inflammation were investigated. METHODS Pulp exposure was made in mandibular right first molars of 5-week-old Wistar rats. After 24 hours, the thalamus was retrieved and subjected to either immunohistochemistry for class II major histocompatibility complex (MHC) molecules and glial fibrillary acidic protein (GFAP) or mRNA expression analysis of antigen-presenting cell-related molecules and N-methyl-D-aspartate receptor 2D subunit (NR2D) by means of reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. RESULTS At 24 hours after pulp exposure, the density of class II MHC molecule-expressing and GFAP-expressing cells was increased in the contralateral thalamus. Gene expression analysis revealed the up-regulation of class II MHC molecules, CD80, CD83, CD86, and NR2D in the contralateral thalamus, as compared with the ipsilateral thalamus. CONCLUSIONS These results suggest the signal of pulp inflammation induces neuronal activation in the CNS.

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.

Increased Gene Expression of Toll-like Receptors and Antigen-Presenting Cell–related Molecules in the Onset of Experimentally Induced Furcation Lesions of Endodontic Origin in Rat Molars

INTRODUCTION Early immunopathogenic mechanisms behind pulp infection-induced furcal inflammation have not been well understood. To address the immunopathology of the pulp infection-induced furcal region of the periodontal ligament (PDL), we performed immunohistochemical and quantitative gene expression analyses for toll-like receptors (TLRs) in the furcal PDL of rat molars subjected to unsealed or sealed pulpotomy. METHODS Furcal inflammation in rat molars was generated by making unsealed pulpotomies that were exposed to the oral environment for 24 hours. Pulpotomized teeth sealed with a temporary filling material and untreated normal teeth served as controls. Gene expression was analyzed with laser capture real-time polymerase chain reaction for TLR-2, TLR-4, and antigen presenting cell (APC)-related molecules (class II MHC, CD83, and CD86). Immunohistochemistry for TLR-2 and TLR-4 was also performed. RESULTS Messenger RNA expression levels of TLRs and the APC-related molecules in the furcal periodontal ligament were significantly up-regulated in teeth with unsealed pulpotomy. Immunohistochemistry for unsealed pulpotomized teeth revealed that TLRs-expressing cells were predominantly distributed within the PDL beneath the furcal dentin. CONCLUSIONS These results suggested the involvement of innate immune mechanisms involving TLRs and resulting activation of APCs in the early pathogenesis of pulp infection-induced furcal inflammation.

Gene expression analysis of acutely traumatized pulps.

INTRODUCTION Vertical root fracture of vital teeth is a relatively rare occurrence. To address early molecular biologic events that take place in the pulp of such cases, we measured mRNA expression levels of selected molecules related to nociception, bacterial pattern recognition, and hard tissue repair/mineralization. METHODS Three extracted roots obtained from vital molars diagnosed as vertical root fracture were used. The samples were first analyzed with light and transmission electron microscopy. Then mRNA expression in the apical (root fractured) and coronal portions of the pulp was analyzed by using reverse transcription-polymerase chain reaction (PCR) or real-time PCR after laser capture microdissection. RESULTS In all the samples, cracks and vital pulp tissue, but not signs of infection and inflammation, were recognized in the apical portion of the fractured root. The gene expression analysis showed that mRNAs of pattern recognition receptors (CD14, Toll-like receptor 2, and Toll-like receptor 4) and neurokinin-1 receptor were equally expressed in both regions of the pulp. On the other hand, mRNA expression levels of hard tissue-associated proteins (osteopontin, osteonectin, and osteocalcin) and calcium channel voltage-dependent alpha 2 delta subunit 1 (CACNA2D1) in the apical portion of the pulp tissue and periodontal ligaments were significantly up-regulated, as compared with those in the coronal portion of the pulp. CONCLUSIONS In the traumatized apical pulp of vertically root-fractured vital teeth, elevated mRNA expression of CACNA2D1, a nociception-related molecule, and proteins related to hard tissue repair/mineralization occurs under noninfectious condition.

Up-regulation of p38 Mitogen-activated Protein Kinase during Pulp Injury–induced Glial Cell/Neuronal Interaction in the Rat Thalamus

INTRODUCTION We have recently reported that the signal of pulp injury induces both neuronal and glial cell activation in the contralateral thalamus in rats, although the mechanisms of the glial cell/neuronal interaction remain unclear. This study was undertaken to test our hypothesis that p38 mitogen-activated protein kinase (MAPK) signaling pathways are involved in the pulp injury-induced glial cell/neuronal interaction in the thalamus. METHODS A local anesthetic (lidocaine with epinephrine) or saline (control) was injected into the tissue surrounding the left mandibular first molar of Wistar rats. The tooth was then pulp-exposed, and the cavity was sealed with flowable composite. After 0 (normal pulp with local anesthetic or saline pretreatment), 24, and 72 hours, the contralateral side of thalamus was retrieved and subjected to immunohistochemistry for phospho-p38 MAPK and glial fibrillary acidic protein and real-time polymerase chain reaction analysis of p38-MAPK family (MAPK 13 and MAPK 14) mRNAs. RESULTS The area immunopositive to phospho-p38 MAPK increased until 72 hours after pulp exposure in both local anesthetic-pretreated and saline-pretreated animals, but the rate of increase was lower in the local anesthetic-pretreated animals. The density of glial fibrillary acidic protein-expressing astrocytes showed a significant increase only in the saline-pretreated animals. Expression levels of MAPK 13 and MAPK 14 mRNAs increased at 24 hours and still higher at 72 hours in the saline-pretreated animals. Notably, MAPK 13 and MAPK 14 mRNA levels at 24 and 72 hours in the local anesthetic-pretreated animals showed significantly lower levels than those in the saline-pretreated animals. CONCLUSIONS It was concluded that pulp injury-induced up-regulation of MAPK 13, MAPK 14, and phospho-p38 MAPK in the thalamus was suppressed by the local anesthetic pretreatment, suggesting the involvement of p38 MAPK signaling pathways in the glial cell-neuronal interaction induced by pulpal nociception.

Observation of the pulp horn by swept source optical coherence tomography and cone beam computed tomography

Cone-beam computed tomography (CBCT) is one of the most useful diagnostic techniques in dentistry but it involves ionizing radiation, while swept source optical coherence tomography (SS-OCT) has been introduced recently as a nondestructive, real-time, high resolution imaging technique using low-coherence interferometry, which involves no ionizing radiation. The purpose of this study was to evaluate the ability of SS-OCT to detect the pulp horn (PH) in comparison with that of CBCT. Ten extracted human mandibular molars were used. After horizontally removing a half of the tooth crown, the distance from the cut dentin surface to PH was measured using microfocus computed tomography (Micro CT) (SL) as the gold standard, by CBCT (CL) and by SS-OCT (OL). In the SS-OCT images, only when PH was observed beneath the overlying dentin, the distance from the cut dentin surface to PH was recorded. If the pulp was exposed, it was defined as pulp exposure (PE). The results obtained by the above three methods were statistically analyzed by Spearmans rank correlation coefficient at a significance level of p < 0.01. SS-OCT detected the presence of PH when the distance from the cut dentin surface to PH determined by SL was 2.33 mm or less. Strong correlations of the measured values were found between SL and CL (r=0.87), SL and OL (r=0.96), and CL and OL (r=0.86). The results showed that SS-OCT images correlated closely with CBCT images, suggesting that SS-OCT can be a useful tool for the detection of PH.

Apices of maxillary premolars observed by swept source optical coherence tomography

Apicoectomy is performed for the management of apical periodontitis when orthograde root canal treatment is not possible or is ineffective. Prior to the surgery, cone beam computed tomography (CBCT) examination is often performed to evaluate the lesion and the adjacent tissues. During the surgical procedure, the root apex is resected and the resected surface is usually observed under dental operating microscope (DOM). However, it is difficult to evaluate the details and the subsurface structure of the root using CBCT and DOM. A new diagnostic system, swept source optical coherence tomography (SS-OCT), has been developed to observe the subsurface anatomical structure. The aim of this study was to observe resected apical root canals of human maxillary premolars using SS-OCT and compare the findings with those observed using CBCT and DOM. Six extracted human maxillary premolars were used. After microfocus computed tomography (Micro CT; for gold standard) and CBCT scanning of the root, 1 mm of the apex was cut perpendicular to the long axis of the tooth. Each resected surface was treated with EDTA, irrigated with saline solution, and stained with methylene blue dye. The resected surface was observed with DOM and SS-OCT. This sequence was repeated three times. The number of root canals was counted and statistically evaluated. There was no significant difference in the accuracy of detecting root canals among CBCT, DOM and SS-OCT (p > 0.05, Wilcoxon test). Because SS-OCT can be used in real time during surgery, it would be a useful tool for observing resected apical root canals.