1 Volume 24 2025 e258423 Original Research Braz J Oral Sci. 2025;24:e258423http://dx.doi.org/10.20396/bjos.v24i00.8678423 1 Division of Cariology and Restorative Dentistry, São Leopoldo Mandic Dental Institute, Campinas, SP, Brazil. 2 Laboratory of Neuroimmune Interface of Pain Research, São Leopoldo Mandic Dental Institute, Campinas, SP, Brazil. 3 Division of Cariology and Restorative Dentistry, São Leopoldo Mandic Dental Institute, Campinas, SP, Brazil. Corresponding author: Cecilia Pedroso Turssi Instituto e Centro de Pesquisas Odontológicas São Leopoldo Mandic Rua José Rocha Junqueira, 13 - CEP 13045-755 Campinas, SP, BRAZIL Telephone: +55-19-3211-3600; Fax: +55-19-3211-3712 email: cecilia.turssi@gmail.com Editor: Dr. Altair A. Del Bel Cury Received: November 18, 2024 Accepted: December 04, 2024 Dentin hypersensitivity: expression of neuron/odontoblast receptors and release of neuropeptide in dental pulp Giovanna Corrêa Denucci1 , Henrique Ballassini Abdalla2 , Juliana Trindade Clemente-Napimoga2 , Cecilia Pedroso Turssi3* Aim: This study assessed the activity of neuron/odontoblast receptors and neuropeptide release, under an animal model of dentin hypersensitivity (DH). Methods: Wistar rats were allocated in two groups (n=10): in test group DH was induced by a validated protocol in which a sports drink (pH 3.08) was ingested for 45d, while in control group, the animals ingested filtered water instead. Animals were euthanized and blood samples were collected to measure plasma corticosterone levels. Dental pulp samples (n=6) were processed for Western Blot and ELISA analysis of TRPV1 (mechano-, thermo- and chemoreceptor), P2X7 (adenosine triphosphate (ATP)-mechanosensitive receptor), TRPM8 (cold-sensitive receptor) and substance P (neurogenic peptide released by neuron activation and due to TRPV1 expression). Data were analyzed using Student’s t tests (α=0.05). Results: DH significantly increased expression of TRPV1 (p=0.002), P2X7 (p=0.007) and substance P (p<0.001) but did not significantly affect the activity of TRPM8 (p=0.079). Conclusion: Under DH condition, neurons and odontoblasts expressed TRPV1 and P2X7 receptors as well as increased substance P release, demonstrating cellular and molecular mechanisms underlying DH. Keywords: Dentin sensitivity. Neurons. Odontoblasts. Neurogenic Inflammation. https://orcid.org/0000-0002-6970-0377 https://orcid.org/0000-0002-7517-2830 https://orcid.org/0000-0003-1068-3039 https://orcid.org/0000-0002-0078-9895 2 Denucci et al. Braz J Oral Sci. 2025;24:e258423 Introduction Dentin hypersensitivity (DH) is a prevalent finding occurring approximately in 33.5% of the population according to a metanalytic study1. DH is described as a short, sharp pain, in response to thermal, evaporative, tactile, osmotic or chemical stimuli after exclusion of other pathologies2,3. For such, it is necessary that dentine becomes exposed to the oral environment, which occurs with gingival recession or loss of enamel due to wear processes, mainly erosion and abrasion4. The main hypotheses that explain DH are threefold: hydrodynamic, dentine innervation and odontoblastic theories5-7. The first, and most accepted attributes pain to the fluid flow within dentinal tubules, stimulating, by pressure difference, nerve endings. On the other hand, the innervation theory states that DH is due to the direct stimulation of nerve endings that extend from the pulp and are present in dentine. The odontoblastic theory suggests that odontoblasts act as receptor cells detecting diverse stimuli. Dental pulp is innervated by two types of nerve fibers, A-fibers (myelinated) and C-fi- bers (non-myelinated)8. Acute pain, which characterizes DH, is related to the activa- tion of the intradental A-fibers5,9. Functionally, A-fibers respond to several stimuli that induce intradentinal fluid flow, being associated to DH by the hydrodynamic theory10. This is due to the fact that increased intratubular fluid flow causes pressure change that consequently activates and stimulates A-fibers at the pulp-dentine interface by means of mechanoreceptors that activate pulp nerves11. Although over time of dentine exposure A-fibers may become less sensitive to hydro- dynamic stimulation, nerve sensitization may persist triggered by inflammatory reactions (activation of C-fibers)12. That is the reason why DH has been considered a chronic state of pain by some researchers10,12. Although literature suggests that patients with DH can have severe pain after initial stimulation followed by lingering pain9, the neurochemical mechanisms that involve potential pulp changes of teeth with DH remain unclear5,13. C-fibers do not respond directly to fluid flow stimuli, but their function in DH may be important because such fibers contain neuropeptides such as substance P and CGRP (calcitonin gene-related peptide), which cause local vasodilation, vascular permea- bilization and edema formation, known as neurogenic inflammation14. In fact, while evaluating the photobiomodulation by low-level laser as a treatment for DH, a paper showed increased expression of substance P in thermal-sensitive dentine15. Besides the release of substance P and CGRP by C-fibers, such neuropeptides can also be secreted following nerve sensitization of inflammatory receptors expressed in odontoblasts10. These ion channels include TRPV1, a member of the transient recep- tor potential (TRP) family of cation channels10,16,17, that acts as mechano-, thermo- and chemosensors5. Neurons and odontoblasts can express indeed TRPM8, a cold-sensing ion channel receptor10,16,17. TRPM8 expression was detected in 58% of sensory neurons innervat- ing the dental pulp of rats18, and in cultured odontoblasts from rat and human dental pulp19. 3 Denucci et al. Braz J Oral Sci. 2025;24:e258423 P2X7 is another odontoblast receptor20, which participates of the sensory function of teeth by releasing extracellular adenosine triphosphate (ATP) in response to phys- ical stimuli21,22. Once activated by P2X7, inflammatory cells release pro-inflammatory cytokines that initiate and maintain a persistent inflammatory process23. However, to best authors’ knowledge, the expression of P2X7 from the DH perspective remains unexplored. Owing to the putative expression of neuron and odontoblast receptors and the still debate around the release of neuropeptides in the context of DH and ultimately the importance of deepening the understanding on its physiopathology under a validated animal model of DH, this study assessed the activity of mechano-, thermo- and/or chemoreceptors as well as of mechanoreceptive ion channel in neurons and odonto- blasts. The null hypothesis tested was that DH would not be associated with activity of receptors in neurons and odontoblasts and neuropeptide release. Materials & Methods Ethical aspects, experimental design and sample size All procedures performed in this study were in accordance with the ethical standards in compliance with the guidelines of the National Council for Animal Experimentation Control (CONCEA) and Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for the care and welfare of animals. This study was reviewed and approved by the local Ethics Committee on Animal Experimentation (protocol #2017/029). Twenty male Wistar rats (ANILAB) were housed in groups of two, in polypropylene cages containing pine shavings in an open system, ventilated shelves with tem- perature ranging between 22 and 24 °C in the animal facility, in an environment with luminosity control (12h light/dark cycles). The animals were randomly assigned to two groups (test and control), as follows: test group: ten rats received a sports drink (Gatorade®, pH 3.08) ad libitum, for 45 days, to induce HD as described elsewhere24,25; control group: ten rats received filtered water (pH 6.30) ad libitum, for 45 days. Both groups received the same feed (Presence Ratos e Camundongos, Presence Nutrição Animal) ad libitum. All animals were weighed before the commencement and at the end of the experiment, for control and certification of welfare. Sample size was calculated (G*Power 3.1.9.4) from the data collected in pilot study and determined that 6 rats per group would provide 90% power to detect an effect size of 2.317 with a 2-sided α of 0.05. Four animals were added in order to provide material for scanning electron microscopy (SEM) analysis and to compensate for dropouts/ losses. Sample collection and processing At the end of the 45-day period of ingestion of the beverages, the rats were euth- anized by decapitation without previous anesthesia to avoid anesthetic-induced increase in plasma corticosterone concentrations26. Blood aliquot was collected in heparin-coated tubes and centrifuged (10 min/1000 ×g). Plasma was used to deter- mine corticosterone level by enzyme-linked immunosorbent assay (ELISA). 4 Denucci et al. Braz J Oral Sci. 2025;24:e258423 Subsequently, the teeth (lower molars) were carefully extracted to prevent dental pulp exposure. Teeth were gently cleaned with sterile gauze and physiological solution, aiming to remove any retained periodontal tissue. They were then stored at -80°C until further analysis. Each sample was composed of a pool of all lower molars (6 teeth). Dental pulp was extracted by triturating the samples in a mortar and pestle. During the extraction process, samples were kept in nitrogen solution avoiding pro- tein denaturation. Samples were then resuspended in RIPA Lysis Buffer (Santa Cruz Biotechnology) containing a protease inhibitor cocktail (Sigma, USA). The homoge- nized samples were centrifuged (10,000 rpm/10 min/4°C), and the supernatant was collected. Homogenized samples were stored at -20°C. Western Blot Total extracted proteins were evaluated using colorimetric BCA protein dosing kit (Thermo Scientific). Protein samples (80 µg) from lower molars were separated by electrophoresis in polyacrylamide gel SDS-PAGE 10% and transferred to nitrocellu- lose membranes. Membranes were incubated overnight at 4 °C with a blocking buf- fer [PBS 5% (p/v) of skim milk and 0,1% Tween 20 and rinsed three times with PBS 0,1% Tween 20. Afterwards, they were incubated in a PBS solution containing 5% skim milk and 0,1% Tween 20 containing primary antibody for anti-rat TRPV1 (Alomone Labs, CAT# ACC029, LOT# ACC030AN2302, 1:1000 overnight), anti-TRPM8 (Alomone Labs, CAT# ACC-049, LOT# ACC049AN1402, 1:500 overnight), and anti-P2X7 (Alo- mone Labs, CAT# APR-004, LOT# APR004AN2302, 1:500 overnight). After washing, the membranes were incubated with specific HRP conjugated secondary antibod- ies and rinsed again. Membranes were then developed with a chemiluminescent kit as described in the instruction manual. Image J software was used to measure the bands by optical density to quantify the protein level of the TRPV1, TRPM8 and P2X7 receptors in dental pulp. To compensate for any differences in the amount of loaded protein, the intensity of the test band was divided by the intensity of GAPDH (Cell Sig- naling Technology®, Denver, MA, USA) band for each sample. Enzyme-linked immunosorbent assay (ELISA) Protein levels of substance P (SP) in dental pulp (Phoenix Pharmaceuticals®, Inc., Burlingame, California, EUA – range 0.07 - 2.24 ng/ml) and corticosterone dosage in plasma (Cayman Chemical® – Ann Arbor, MI, USA – range 8.2 – 5,000 pg/ml) were evaluated by ELISA according to manufacturer’s specifications of the kits used. SEM The hemimandibles of the remaining four animals of each group were extracted and stored in formaldehyde. Subsequently, the soft tissue was removed and the hemiman- dibles underwent dehydration with ethyl alcohol, in increasing concentrations: 25% (20 minutes), 50% (20 minutes), 75% (20 minutes), 95% (30 minutes) and absolute alcohol (100%) for 1 hour. Then, hexamethyldisilane (HMDS) was used for 10 minutes to dry the samples and a layer of gold (200 Aº) was deposited on them (Sputter Coater EMITECH, Model: K450. Kent, United Kingdom) to be analyzed in a scanning electron 5 Denucci et al. Braz J Oral Sci. 2025;24:e258423 microscope (Leo 440i, LEO Electron Microscopy/Oxford. Cambridge, England). Photo- micrographs were obtained at 80x, 500x and 5,000x magnification. Statistical analysis After ascertaining data compliance to normal distribution and homoscedasticity, the groups were compared using Student’s t tests (GraphPad Prism, GraphPad software Inc, California, USA), with the significance level set at 0.05. Results SEM photomicrographs substantiated the exposure and patency of dentinal tubules in test animals, i.e., that received the sports drink (Figures 1a-f). The top row are images from the control group, and the bottom row images are from the test group. The first column (A and D) are zoomed-out images (50x magnification) that show the buccal and occlusal surfaces of the molars, it is possible to notice the amount of enamel loss and dentine exposure on SEM image D, while the dentine in the control group is not exposed. The second column (B and E) are a 500x magnification of the cervical area, again the control group has enamel, while the test group image shows the amount of enamel loss, dentinal exposure, and dentinal tubules are visible. The last column (C&F) are 5,000X magnification show enamel in the control group and exposed and open dentinal tubules in the test group. A B C D E F Legend: Top row images a, b and c from the control group; Bottom row images d, e and f from the test group; DE- Dental enamel, PB- Periodontal bone, ED- Exposed dentine in areas of enamel loss, DT- Dentinal tubule. Figure 1. Scanning electron microscopy (SEM) photomicrographs of rat teeth from control and test groups. 6 Denucci et al. Braz J Oral Sci. 2025;24:e258423 The test group demonstrated significantly higher levels of TRPV1 (p = 0.002; Figure 2A) and P2X7 receptors (p = 0.007; Figure 2B) and substance P (p < 0.001; Figure 2C). However, there was no difference in the level of TRPM8 between groups (p = 0.079; Figure 2D). Control Control ControlTest Test Control Test Control Test Control Test Test Control Test TRPV1 P2X7R TRPV1 – 98 KDa GAPDH – 35 KDa P2X7 – 75 KDa GAPDH – 35 KDa TRPM8 – 130 KDa GAPDH – 35 KDa ** *** ** 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 0.6 0.4 0.2 0.0 1.5 1.0 0.5 0.0 Fo ld c ha ng e pu lp ti ss ue Fo ld c ha ng e pu lp ti ss ue Pr ot ei n le ve l ( ng /m L) pu lp ti ss ue Fo ld c ha ng e pu lp ti ss ue TRPM8Substance P ns A B C D Legend: The symbol (***) indicates p < 0.001 and (**) indicates p < 0.01. Figure 2. Column diagram of the mean expression of TRPV1 (A), P2X7 (B), Substance P (C), and TRPM8 (D) in dental pulp of the rats of control and test groups. Corticosterone level was significantly higher in the test group (p = 0.050; Figure 3), while there was no difference in the body weight of the rats in the two groups (p = 0.756; Table 1). 7 Denucci et al. Braz J Oral Sci. 2025;24:e258423 Control Test Corticosterone **250 200 150 100 50 0 Pr ot ei n le ve l ( pg /m L) Bl oo d Se ru m Legend: The symbol (**) indicates p = 0.05, Student’s t test. Figure 3. Column diagram of the mean plasma corticosterone level of the control and test groups. Table 1. Average and standard deviation of the initial and final weight (g) of the control and test animals. Time Control Test Initial 332.8 (12.8) 331.6 (10.4) Final 399.3 (21.6)a 402.6 (13.2)a Legend: Final weight followed by the same lowercases did not differ from each other. Discussion The rat DH model used in this study has proved to cause substantial surface loss of dental hard tissues, exposure and patency of dentinal tubules (Figure 1), in alignment with papers that validated this method of inducing hypersensitive dentine24,25. With respect to this model, it is worth noting that in a previous paper by our group27, despite the severity of the erosion lesions, no histological changes such as inflammation and reparative dentin formation have been noticed in the underlying pulp tissue. In addition to structural and morphological changes on the dental hard tissues, it is worth noting that our results showed a more than threefold increase in plasma corticosterone levels in the group with DH. This allows inferring that there was pain associated with the wear lesions, a finding consistent with the fact that most of the animals subjected to the same DH model presented high scores of pain in previous investigations24,25. Although one can argue that corticosterone levels was increased as a consequence of handling, all animals was habituated to daily manipulation and fast handled for similar time prior to euthanasia, which was performed by decapita- tion without anesthesia for data quality. Despite the severity of surface loss, dentine exposure and pain in the test group, which could imply limitations in feeding, animals of both groups had no weight differences. Based on the findings of this paper, the null hypothesis that HD would not be associ- ated with activity of receptors in neurons and odontoblasts and neuropeptide release was rejected as DH significantly increased expression of TRPV1, P2X7 and substance P under the DH animal model adopted herein. 8 Denucci et al. Braz J Oral Sci. 2025;24:e258423 The higher TRPV1 expression in the group having hypersensitive dentine gives evi- dence to previous conjectures that the experience of pain via exposed dentine may involve the activation of TRPs17. According to the quoted paper, such expression relies on receptors present in both nerve fibers and odontoblasts. As in the current study dental pulp was extracted from triturated lower molars, the origin of TRPV1 (if from nerve fibers or odontoblasts) cannot be differentiated. However, in the clini- cal setting probably the most important expression of TRPV1 would originate from odontoblasts, which extents up to inner dentine from pulp28, and thereby represents the outermost cellular component that external stimuli would encounter in tooth structure. On the other hand, nerve fibers possibly would have a secondary role, as they are present in dentinal tubules only approximately 100 μm from the pulp29. This discussion is in line with a previous paper in which the authors mentioned that TRPV1 is detected mainly in odontoblasts16. Activation of TRPV1 has been shown to promote release of substance P10, caus- ing neurogenic inflammation through vasodilation, vascular permeabilization and edema formation10. It seems therefore reasonable to consider that the increased expression of TRPV1 and substance P release in DH context, as it occurs in pulp from carious teeth30 and teeth diagnosed with irreversible pulpitis31, has the poten- tial to cause neurogenic inflammation and evoke pain. Worth mentioning is that once substance P is elevated, it is further increased during the inflammatory pro- cess, thereby sustaining, and exacerbating the neurogenic inflammation14, which may explain DH as a lingering pain in some patients and even help to explain the eventual need of endodontic treatment for irreversible pulpitis of teeth having non-carious cervical lesions. Under the current model, expression of the P2X7 receptor in the pulp was also higher in the test group, providing evidence on the sensory role of odontoblasts under a DH scenario. This role can be ascribed to the increased TRPV1 expression that induces ATP release to the extracellular space by stimulated odontoblasts32. In fact, membrane deformation caused by dentinal fluid movement activates mechanosensitive channels-TRP33. ATP is then released into the extracellular space, acting as a neurotransmitter, and activates P2X7 receptors, which trigger intracellular signaling pathways thereby activating the neuron to establish neuro- transmission with neurons33. Despite the borderline p value suggests a trend toward pulp samples of rats having DH present increased TRMP8 activity, no statistically significant difference was detected among the test and control groups. One possible reason for this finding may be the fact that TRPM8 starts to be sensitive to cooling approximately below 25 °C and 22 °C in dental pulp nerves34 and odontoblasts35, respectively, and the sports drink was avail- able and consumed at temperature close to this threshold (22-24 °C). One can specu- late that lower temperatures, similar to the temperature that the drink is consumed in real life situations, neurons and odontoblasts would have increased response, thereby elevating calcium ions and TRPM8 activity17. Although there exist other neurogenic peptides and numerous classes of mechano- and thermosensitive channels as well as ligand- and voltage-gated ion channels, their roles when dentine is exposed remain to be unraveled. Yet, by assessing some 9 Denucci et al. Braz J Oral Sci. 2025;24:e258423 of the main neuron and odontoblast receptors (TRPV1, TRPM8 and P2X7) and neu- rogenic peptide (substance P) under an animal model of DH, this paper contributes to the comprehension on the physiopathology and advancing in strategies to pre- vent and treat DH. Despite further studies are warranted to perform immunohisto- chemical to deepen the comprehension of the cellular expression of the receptors, the present investigation opens perspectives to advance strategies to prevent and treat DH. This may include the incorporation of pharmacological antagonists of neu- ron and odontoblast receptors to dentine desensitizing formulations. In conclusion, under DH condition, neurons and odontoblasts expressed TRPV1 and P2X7 receptors as well as increased substance P release, demonstrating cellular and molecular mechanisms underlying DH. Acknowledgements To the Coordination for the Improvement of Higher Education Personnel for pro- viding access to scientific journals online. This work was supported by grants from São Paulo Research Foundation (FAPESP, grants #2016/16529-9, #2017/22334-9), National Council for Scientific and Technological Development (CNPq) [Research Pro- ductivity Fellowship to JTCN]. Data Availability Datasets related to this article will be available upon request to the corresponding author. Author Contributions Giovanna Correa Denucci: conceived the idea, designed the experiment, col- lected data, analyzed data, wrote and revised the manuscript. Henrique Ballassini Abdalla: collected data, analyzed data, revised the manuscript. Juliana Trindade Clemente-Napimoga: conceived the idea, designed the experiment, analyzed data, revised the manuscript. Cecilia Pedroso Turssi: conceived the idea, designed the experiment, analyzed data, revised the manuscript. References 1. Favaro Zeola L, Soares PV, Cunha-Cruz J. Prevalence of dentin hypersensitivity: systematic review and meta-analysis. J Dent. 2019 Feb;81:1-6. doi: 10.1016/j.jdent.2018.12.015. 2. Dowell P, Addy M. Dentine hypersensitivity - a review. Aetiology, symptoms and theories of pain production. J Clin Periodontol. 1983 Jul;10(4):341-50. doi: 10.1111/j.1600-051x.1983.tb01283.x. 3. Pashley DH. How can sensitive dentine become hypersensitive and can it be reversed? J Dent. 2013 Jul;41 Suppl 4:S49-55. doi: 10.1016/S0300-5712(13)70006-X. 4. West N, Seong J, Davies M. Dentine hypersensitivity. Monogr Oral Sci 2014;25:108-22. doi: 10.1159/000360749. 5. Solé-Magdalena A, Martínez-Alonso M, Coronado CA, Junquera LM, Coboc J, Vega JA. Molecular basis of dental sensitivity: the odontoblasts are multisensory cells and express multifunctional ion channels. Ann Anat. 2018 Jan;215:20-9. doi: 10.1016/j.aanat.2017.09.006. Epub 2017 Sep 24. 10 Denucci et al. Braz J Oral Sci. 2025;24:e258423 6. Won J, Oh SB. Update on dentin hypersensitivity: with the focus on hydrodynamic theory and mechanosensitive ion channels. Int J Oral Biol. 2019;44(3):71-6. doi: 10.11620/IJOB.2019.44.3.71. 7. Aminoshariae A, Kulild JC. Current concepts of dentinal hypersensitivity. J Endod. 2021 Nov;47(11):1696-702. doi: 10.1016/j.joen.2021.07.011. 8. Nair PN. Neural elements in dental pulp and dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1995 Dec;80(6):710-9. doi: 10.1016/s1079-2104(05)80256-2. 9. Närhi M, Jyväsjärvi E, Virtanen A, Huopaniemi T, Ngassapa D, Hirvonen T. Role of intradental A- and C- type fibres in dental pain mechanisms. Proc Finn Dent Soc. 1992;88 Suppl 1:507-16. 10. Le Fur-Bonnabesse A, Bodéré C, Hélou C, Chevalier V, Goulet JP. Dental pain induced by an ambient thermal differential: pathophysiological hypothesis. J Pain Res. 2017 Dec;10:2845-51. doi: 10.2147/JPR.S142539. 11. Brännström M, Johnson G. Movements of the dentine and pulp liquids on application of thermal stimuli. An in vitro study. Acta Odontol Scand. 1970 Mar;28(1):59-70. doi: 10.3109/00016357009033132. 12. Närhi M, Yamamoto H, Ngassapa D, Hirvonen T. The neurophysiological basis and the role of inflammatory reactions in dentin hypersensitivity. Arch Oral Biol. 1994;39 Suppl:23S-30S. doi: 10.1016/0003-9969(94)90184-8. 13. Lee K, Lee BM, Park CK, Kim YH, Chung G. Ion channels involved in tooth pain. Int J Mol Sci. 2019 May 8;20(9):2266. doi: 10.3390/ijms20092266. 14. Sacerdote P, Levrini L. Peripheral mechanisms of dental pain: the role of substance P. Mediators Inflamm. 2012;2012:951920. doi: 10.1155/2012/951920. 15. de Oliveira VT, Ferrara-Jr JI, Matielo HA, da Silva Alves A, Britto LR, Aranha ACC, et al. Involvement of substance P, osteopontin and satellite glial cells on photobiomodulation-induced antinociceptive effect in an experimental model of dentin hypersensitivity. Lasers Med Sci. 2021 Aug;36(6):1297-305. doi: 10.1007/s10103-021-03246-9. 16. El Karim IA, Linden GJ, Curtis TM, About I, McGahon MK, Irwin CR, et al. Human odontoblasts express functional thermo-sensitive TRP channels: implications for dentin sensitivity. Pain. 2011 Oct;152(10):2211-23. doi: 10.1016/j.pain.2010.10.016. Epub 2010 Dec 17. 17. Hossain MZ, Bakri MM, Yahya F, Ando H, Unno S, Kitagawa J. The role of Transient Receptor Potential (TRP) channels in the transduction of dental pain. Int J Mol Sci. 2019 Jan;20(3):526. doi: 10.3390/ijms20030526. 18. Kim YS, Kim YJ, Paik SK, Cho YS, Kwon TG, Ahn DK, et al. Expression of metabotropic glutamate receptor mGluR5 in human dental pulp. J Endod. 2009 May;35(5):690-4. doi: 10.1016/j.joen.2009.02.005. 19. Tokuda M, Tatsuyama S, Fujisawa M, Morimoto-Yamashita Y, Kawakami Y, Shibukawa Y, et al. Dentin and pulp sense cold stimulus. Med Hypotheses. 2015 May;84(5):442-4. doi: 10.1016/j.mehy.2015.01.039. 20. Lee BM, Jo H, Park G, Kim YH, Park CK, Jung SJ, et al. Extracellular ATP induces calcium signaling in odontoblasts. J Dent Res. 2017 Feb;96(2):200-7. doi: 10.1177/0022034516671308. Epub 2016 Oct 2. 21. Liu X, Wang C, Fujita T, Malmstrom HS, Nedergaard M, Ren YF, et al. External dentin stimulation induces ATP release in human teeth. J Dent Res. J Dent Res. 2015 Sep;94(9):1259-66. doi: 10.1177/0022034515592858. 22. Sato M, Furuya T, Kimura M, Kojima Y, Tazaki M, Sato T, et al. Intercellular odontoblast communication via ATP mediated by pannexin-1 channel and phospholipase C-coupled receptor activation. Front Physiol. 2015 Nov;6:326. doi: 10.3389/fphys.2015.00326. 11 Denucci et al. Braz J Oral Sci. 2025;24:e258423 23. North RA. P2X receptors. Philos Trans R Soc Lond B Biol Sci. 2016 Aug;371(1700):20150427. doi: 10.1098/rstb.2015.0427. 24. Bergamini MR, Bernardi MM, Sufredini IB, Ciaramicoli MT, Kodama RM, Kabadayan F, et al. Dentin hypersensitivity induces anxiety and increases corticosterone serum levels in rats. Life Sci. 2014 Mar;98(2):96-102. doi: 10.1016/j.lfs.2014.01.004. 25. Braga TM, Braga DN, Moreno-Carvalho E, Bauer JO, Turssi CP. Calcium pre-rinse: effect on permeability of dentin tubules by fluoride rinse. 2019 Apr;11(4):e303-9. doi: 10.4317/jced.55382. 26. Barbosa FM, Cabral D, Kabadayan F, Bondan EF, de Fátima Monteiro Martins M, Kirsten TB, et al. Depressive behavior induced by unpredictable chronic mild stress increases dentin hypersensitivity in rats. Arch Oral Biol. 2017 Aug;80:164-74. doi: 10.1016/j.archoralbio.2017.04.005. 27. Fagundes-de-Souza DP, Napimoga MH, Soares AB, Araújo VC, Turssi CP. Does hypersensitive teeth show pulp inflammation? Rev Gaucha Odontol. 2019;67:e20190011. doi: 10.1590/1981-86372019000113580. 28. Sigal MJ, Aubin JE, Ten Cate AR, Pitaru S. The odontoblast process extends to the dentinoenamel junction: an immunocytochemical study of rat dentine. J Histochem Cytochem. 1984 Aug;32(8):872-7. doi: 10.1177/32.8.6379038. 29. Brännström M. The hydrodynamic theory of dentinal pain:sensation in preparations, caries, and the dentinal crack syndrome. J Endod. 1986 Oct;12(10):453-7. doi: 10.1016/S0099-2399(86)80198-4. 30. Morgan CR, Rodd HD, Clayton N, Davis JB, Boissonade FM. Vanilloid receptor 1 expression in human tooth pulp in relation to caries and pain. J Orofac Pain. 2005 Summer;19(3):248-60. 31. Caviedes-Bucheli J, Gutierrez-Guerra JE, Salazar F, Pichardo D, Moreno GC, Munoz HR. Substance P receptor expression in healthy and inflamed human pulp tissue. Int Endod J. 2007 Feb;40(2):106-11. doi: 10.1111/j.1365-2591.2006.01189.x. 32. Bartlett R, Stokes L, Sluyter R. The P2X7 receptor channel: recent developments and the use of P2X7 antagonists in models of disease. Pharmacol Rev. 2014 Jul;66(3):638-75. doi: 10.1124/pr.113.008003. 33. Shiozaki Y, Sato M, Kimura M, Sato T, Tazaki M, Shibukawa Y. Ionotropic P2X ATP receptor channels mediate purinergic signaling in mouse odontoblasts. Front Physiol. 2017 Jan;8:3. doi: 10.3389/fphys.2017.00003. 34. Park CK, Kim MS, Fang Z, Li HY, Jung SJ, Choi SY, et al. Functional expression of thermo-transient receptor potential channels in dental primary afferent neurons: implication for tooth pain. J Biol Chem. 2006 Jun;281(25):17304-11. doi: 10.1074/jbc.M511072200. 35. Tsumura M, Sobhan U, Sato M, Shimada M, Nishiyama A, Kawaguchi A, et al. Functional expression of TRPM8 and TRPA1 channels in rat odontoblasts. PLoS One. 2013 Dec;8(12):e82233. doi: 10.1371/journal.pone.0082233.