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Expression of Transient Receptor Potential Ankyrin 1 in Human Dental Pulp
Clinical Research
Expression of Transient Receptor Potential Ankyrin 1 in Human Dental Pulp Yun Sook Kim, PhD,* Hoon Kap Jung, DDS,* Tae Kyung Kwon, DDS,† Chin Soo Kim, DDS, PhD,* Jin Hyun Cho, DDS, PhD,* Dong Kuk Ahn, DDS, PhD,* and Yong Chul Bae, DDS, PhD* Abstract Introduction: Transient receptor potential ankyrin 1 (TRPA1) is activated by noxious cold (<17 C) and contributes to cold and mechanical hypersensitivity after inflammation and nerve injury. Methods: To investigate whether TRPA1 is involved in the mediation of nociception, including noxious cold and cold hypersensitivity in teeth, we examined the expression of TRPA1 and sodium channel Nav1.8 in human dental pulp using fluorescent and electron microscopic immunocytochemistry. Results: TRPA1 was expressed in a large number of axons branching extensively in the peripheral pulp and in a few axons within the nerve bundles in the core of the coronal pulp and in the radicular pulp. Under electron microscopy, TRPA1 immunoreactivity was typically localized near the plasma membrane of unmyelinated axons in the peripheral pulp, suggesting that in these axons it may act as a functional receptor. The proportion of axons expressing TRPA1 in neurofilament 200–positive axons significantly increased in the painful pulp compared with the normal pulp. TRPA1 was also densely expressed in the processes and the cell body of odontoblasts. A large number of axons coexpressed TRPA1 and Nav1.8. Conclusions: These findings support the notion that TRPA1 is involved in the perception of noxious cold and cold hypersensitivity in human dental pulp and that TRPA1-mediated nociception is primarily mediated by axons and odontoblasts in the peripheral pulp. (J Endod 2012;38:1087–1092)
Key Words Cold hypersensitivity, dental pulp, immunohistochemistry, inflammation, Nav1.8, nociception, TRPA1
From the *Department of Oral Anatomy and Neurobiology, School of Dentistry, Kyungpook National University, Daegu, Korea; and †Mir Dental Hospital, Daegu, Korea. Supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2011-0028240). Address requests for reprints to Dr Yong Chul Bae, Department of Oral Anatomy and Neurobiology, School of Dentistry, Kyungpook National University, 188-1, 2-Ga, Samdeok-Dong, Jung-Gu, Daegu 700-412, South Korea. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2012 American Association of Endodontists. doi:10.1016/j.joen.2012.04.024
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ental cold hypersensitivity is one of the most frequently suffered symptoms in many adult dental patients. Yet, our knowledge of the mechanisms of dental cold nociception and cold hypersensitivity are just now beginning to be elucidated. Recently, a family of nonselective cation channels (transient receptor potential [TRP] channels) has been shown to render neurons and other cells that express them on their plasma membrane thermosensitive in a fairly narrow and partially overlapping temperature range. Different TRP channels also mediate the action of a variety of pungent compounds in plant extracts on sensory neurons that is perceived as ‘‘hot’’ (eg, capsaicin via the transient receptor potential vanilloid 1) or ‘‘cold’’ (eg, menthol via the transient receptor potential ankyrin 1 [TRPA1]). TRPA1 is expressed primarily in nociceptive neurons and plays a crucial role in the perception of noxious cold (1). It also mediates the hypersensitivity to cold after inflammation and nerve injury (2–4). Recently, it has been shown that neurons in the rat trigeminal ganglion (TG) that innervate the dental pulp express messenger RNA for TRPA1 and that stimulation with noxious cold (<17 C) causes increased intracellular calcium and evoked currents in these neurons (5, 6), implicating TRPA1 in the mediation of noxious cold within the dental pulp. Large numbers of TRPA1-positive (+) axons also terminate in the dorsal part of the trigeminal sensory nuclei that receive intraoral input (7), suggesting that the oral cavity may be densely innervated by TRPA1+ sensory neurons. In addition, a potential role for TRPA1 in human odontoblasts is now beginning to be elucidated (8). However, evidence for a TRPA1-mediated nociception in the dental pulp is still lacking. Voltage-gated sodium channels (Navs) play a crucial role in the generation and propagation of action potentials in neurons. Among the 9 subtypes of Navs in humans, the tetrodotoxin-resistant Nav1.8 is expressed mainly in small-sized nociceptive neurons in the TG (9, 10) and dorsal root ganglia (DRG) (11, 12). Nav1.8-null mice show a negligible behavioral response to noxious cold (13, 14), and their DRG neurons are also unexcitable in the cold (15), suggesting that Nav1.8 is important for the generation and propagation of action potentials at low temperatures. Recently, it has also been reported that rat TG neurons that innervate the dental pulp express messenger RNA for both TRPA1 and Nav1.8 and respond to temperatures below 17 C (5, 6). However, whether Nav1.8 is involved in the transduction of TRPA1-mediated nociception in human pulp is currently unknown. To address these issues, we examined the expression of TRPA1 and Nav1.8 in human dental pulp using fluorescent and electron microscopic immunohistochemistry.
Materials and Methods Tissue Preparation This study was approved by the Research and Ethics Committee of Kyungpook National University, Daegu, Korea, and informed consent was obtained from all human subjects who participated in the experimental investigation after the nature of the procedure had been fully explained. Twelve pulps from healthy premolars extracted during the course of orthodontic treatment (males, age range: 17–21 years old) and 3 pulps from a painful premolar and molar diagnosed with irreversible pulpitis (males, age range: 18–23 years old) at Kyungpook National University Hospital and Mir Dental Hospital, Daegu, Korea, were used for this study. Four normal pulps were used for immunofluorescent staining for TRPA1 and/or Nav1.8, 5 normal pulps and 3 painful
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Clinical Research pulps for immunofluorescent staining for TRPA1 and/or neurofilament 200 (NF200), and 3 normal pulps for electron microscopic (EM) immunohistochemistry for TRPA1. Immediately after extraction, teeth were stored in 0.1 mol/L phosphate buffer (PB) (pH = 7.4) at 4 C for 1 to 4 hours. Then, the teeth were cut longitudinally with a watercooled high-speed diamond bur, and the pulps were fixed for 2 hours in 4% (w/v) paraformaldehyde (PFA) in 0.1 mol/L PB (pH = 7.4) for immunofluorescence or in a mixture of 4% PFA and 0.05% glutaraldehyde for EM immunohistochemistry. For immunofluorescence, the pulps were cryoprotected in 30% sucrose in PB overnight at 4 C and cut on a freezing microtome at 40 mm. For EM immunohistochemistry, the pulps were cut on a vibratome at 60 mm and also cryoprotected in 30% sucrose in PB overnight at 4 C.
Immunofluorescence and Analysis for Immunopositive Axons For immunofluorescence, sections were permeabilized with 50% ethanol for 30 minutes to enhance penetration, blocked with 10% normal donkey serum (Jackson ImmunoResearch, West Grove, PA) for 30 minutes to mask secondary antibody binding sites, and incubated overnight in a mixture of rabbit anti-TRPA1 antiserum (1:1,000, RA14135; Neuromics, Edina, MN) and mouse anti-Nav1.8 antiserum (1:100, 75-166; NeuroMab, Davis, CA) or in a mixture of rabbit anti-TRPA1 antiserum and mouse anti-NF200 antiserum (1:200,000, N0142; Sigma-Aldrich, St Louis, MO) in phosphate-buffered saline (PBS, 0.01 mol/L, pH = 7.4). After washing with PBS, the sections were incubated with appropriate secondary antibodies (fluorescence isothiocyanate or Cy3-conjugated antibodies raised in donkey, 1:200 in PBS, Jackson ImmunoResearch) for 2 hours. The sections were mounted on slides and examined under a fluorescent microscope (Zeiss Axioplan 2; Carl Zeiss Inc, Jena, Germany) or a laser confocal microscope (LSM 510 META, Carl Zeiss Inc). The quantitative analysis of TRPA1 expression within NF200-immunopositive axons was performed on 3 to 4 sections from each of the 5 healthy pulps and from each of the 3 painful pulps with irreversible pulpitis. Three to 4 images from each section were captured from similar regions in the central and peripheral portions of the coronal pulp with an Exi digital camera (Q-Imaging Inc, Surrey, CA) using 20 objectives (1,360 1,036 pixels) and analyzed using NIH ImageJ software (available at http://rsb.info.nih.gov/ij/). The threshold level for classifying pixels as immunopositive for TRPA1 and NF200 was set between 100 and 120 gray levels from images with 256 gray levels. The area fraction (%) of immunopositive axons (TRPA1/NF200) was presented as the mean the standard error.
Data of the normal pulp and the painful pulp with irreversible pulpitis were compared using an unpaired Student’s t test. Quantitative assessment of colocalization of TRPA1 with Nav1.8 was performed on 3 images from each of the 16 sections of 4 healthy coronal pulps. The omission of the primary or secondary antibodies or preadsorption of the TRPA1 antibody with a blocking peptide at a final concentration of 100 mg/mL completely abolished the specific immunostaining (Fig. 1).
EM Immunohistochemistry The procedure for pre-embedding immunohistochemistry was performed as described elsewhere (7, 16). Briefly, cryoprotected sections were frozen on dry ice for 20 minutes, thawed in PBS, and treated with 1% sodium borohydride in PBS for 30 minutes. To suppress endogenous peroxidase, sections were incubated with 3% H2O2 for 10 minutes and blocked with 10% normal donkey serum for 30 minutes. After overnight incubation with rabbit anti-TRPA1 antiserum (1:1,000, Neuromics), sections were incubated in biotinylated donkey antirabbit antiserum (1:200, Jackson ImmunoResearch) for 2 hours, rinsed, incubated with ExtrAvidin peroxidase (1:5,000, Sigma-Aldrich) for 1 hour, and visualized with nickel-intensified diaminobenzidine. Sections were then osmicated (1% osmium tetroxide in PB), dehydrated in graded alcohols, and embedded in Durcupan ACM (Fluka, Buchs, Switzerland). Thin sections were cut, mounted on formvar-coated single-slot nickel grids, counterstained with Sato’s lead, and examined on a Hitachi H 7500 electron microscope (Hitachi, Tokyo, Japan).
Results Strong immunofluorescent staining for TRPA1 was observed in axons and axon collaterals in human dental pulp (Fig. 2). A small number of axons within nerve bundles wrapped with connective tissue were TRPA1 positive (+) in the radicular pulp and in the core of the coronal pulp (Fig. 2A and B). In contrast, in the peripheral pulp, TRPA1+ axons branched extensively and formed a network in the vicinity of the cell-rich zone corresponding to the plexus of Raschow in the occlusal and the cervical regions (Fig. 2C and D). TRPA1+ axons ascending toward the dentin between odontoblasts were rarely observed. The proportion of NF200+ axons expressing TRPA1 significantly increased in the painful pulp compared with the normal pulp (P < .05). Immunoreactivity for TRPA1 was more intense in the painful pulp than in the normal pulp (Figs. 3 and 4). Immunoreactivity for TRPA1 was also observed in the odontoblasts and their processes (Fig. 5A–D). A large number of TRPA1+ axons were immunostained
Figure 1. Immunofluorescent staining for (A) TRPA1 in normal human dental pulp is completely abolished by (B) preadsorption with a control peptide, proving the specificity of the TRPA1 antiserum (200, scale bars = 50 mm).
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Figure 2. Immunofluorescent staining for TRPA1 in (A) the radicular portion, (B) the core of the coronal portion, and (C and D) the peripheral portion (C, occlusal region; D, cervical region) of normal human dental pulp. (A and B) Relatively sparse TRPA1+ axons in the nerve bundle issue few axon collaterals in the radicular pulp and the core of the coronal pulp. (C and D) TRPA1+ axons branch extensively and form a network in the (C) occlusal and (D) cervical regions of the peripheral pulp (200, scale bars = 50 mm).
for Nav1.8. Of the 450 TRPA1+ axons examined in the coronal pulp in 16 sections from 4 pulps, 177 (39.3%) were Nav1.8+ (Fig. 6A–C). At the electron microscopic level, TRPA1 immunoreactivity was observed in the form of an electron-dense amorphous product in the axoplasm of both myelinated and unmyelinated axons in the radicular pulp and the central portion of the coronal pulp. However, it was usually
Figure 3. The area fraction (%) of TRPA1+ axons in NF200+ nerve fibers in the normal and painful (irreversible pulpitis) pulp samples. *A significant difference between normal and painful pulp samples (P < .05).
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observed near the axolemma of unmyelinated axons in the peripheral pulp (Fig. 7).
Discussion The main findings of this study are that TRPA1 is densely expressed in the axons and odontoblasts in the peripheral portion of human dental pulp and the expression of TRPA1 is increased in the pulpal axons after pulpal inflammation. In addition, approximately 40% of TRPA1+ axons coexpress the sodium channel Nav1.8. TRPA1 is selectively expressed by small- and medium-sized, nociceptive neurons in the DRG and TG and has been implicated in the mediation of noxious cold (1, 7, 17) and the cold and mechanical hypersensitivity after inflammation (18). Recently, Park et al (5) showed that rat TG neurons innervating the dental pulp express messenger RNA for TRPA1 and that the application of icilin or noxious cold to these neurons increases their intracellular calcium and evoked cationic currents, suggesting that TRPA1 may contribute to dental pain evoked by noxious cold. The fact that a large number of pulpal axons in the present study expressed TRPA1 is consistent with a TRPA1-mediated cold nociception within human dental pulp. TRPA1 was densely expressed in a large number of axons that branched extensively outside the connective tissue sheath in the peripheral pulp and in a few axons within nerve bundles in the core of the coronal pulp and in the radicular pulp. Electron microscopy revealed that TRPA1 is expressed mostly in unmyelinated peripheral axons, in which it was typically localized at the axonal membrane where it could act as a functional receptor. In contrast, in myelinated axons in the core of the coronal pulp and in the radicular pulp, TRPA1 was localized in the axoplasm away from the membrane, possibly representing a pool of receptors that is being transported to the peripheral pulp. These findings suggest that the TRPA1-mediated sensitivity to noxious cold is
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Figure 4. Double immunofluorescent staining for (A and D) TRPA1 and (B and E) NF200 in the peripheral region of (A–C) normal human pulps and (D–F) painful pulps. Numbers of axons showing TRPA1 expression in NF200+ axons increase in the painful pulp compared with the normal pulp (200, scale bars = 50 mm).
Figure 5. Immunofluorescent staining for TRPA1 in the (A–C) occlusal and (D) cervical regions of normal human dental pulp. Odontoblasts show intense immunoreactivity for TRPA1 in their cell bodies and in their processes (arrowheads). An arrow in C points to a TRPA1+ axon beneath the odontoblast layer (400, scale bars = 50 mm).
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Figure 6. Double immunofluorescent staining for (A) TRPA1 and (B) Nav1.8 in normal human dental pulp. (C) Many TRPA1+ axons are Nav1.8-positive (arrowheads) (200, scale bar = 20 mm).
mediated primarily by axons in the peripheral pulp rather than in the core of the coronal pulp or in the radicular pulp. In addition, very few axons passing between odontoblasts and entering dentinal tubules were TRPA1 positive, suggesting that axons in the dentinal tubules either express another TRP receptor or are insensitive to cold. In the present study, the proportion of axons expressing TRPA1 within NF200+ axons significantly increased, suggesting that TRPA1 is involved in cold and mechanical allodynia and/or hyperalgesia after dental pulp inflammation. Several previous studies have proposed that odontoblasts may act as sensory cells because they express mechanosensitive K+ channels (19, 20), L-type Ca2+ channels (21), and thermosensitive TRP channels including TRPA1 and the transient receptor potential vanilloid 1 (22, 23). They also express voltage-gated sodium channels (24, 25) and may thus be electrically excitable and able to generate action potentials (26). TRPA1 is functionally expressed in other nonneuronal cells, including keratinocytes (27), fibroblasts (28), and synoviocytes (29), which may act in concert with sensory afferents to monitor the thermal environment. In agreement with 1 recent report (8), in this study we showed that TRPA1 is expressed by odontoblasts, thus providing morphologic evidence that human odontoblasts may be involved in sensing and
transducing noxious cold information. TRPA1 immunoreactivity was strong in the processes and in the cell bodies of the odontoblasts. Because, as described previously, TRPA1+ axons were rarely seen ascending toward the dentinal tubule between odontoblasts, we speculate that noxious cold may be sensed primarily by odontoblast processes rather than by axons in the dentinal tubule. About 40% of TRPA1+ axons coexpressed Nav1.8 in this study, corroborating previous reports that TG neurons that innervate the dental pulp express messenger RNA for both TRPA1 and Nav1.8 (5, 6) and that DRG neurons of Nav1.8-null mice have significantly attenuated the expression of TRPA1 (15). Considering that among the 9 subtypes of Nav channels in humans, Nav1.8 can generate an electrical impulse and is involved in the transmission of nociceptive information under cold conditions and plays a crucial role in pain perception at a low temperature (14), our finding supports the notion that Nav1.8 is involved in the conduction of TRPA1mediated cold nociception in human dental pulp. The TRPA1+ axons in the dental pulp that do not express Nav1.8 may express other Navs, possibly Nav1.7 or Nav1.9, both of which have been shown to be overexpressed after pulpal inflammation and have been implicated in the perception of pain after inflammation and nerve injury (30–32).
Figure 7. Electron micrographs showing immunoperoxidase staining for TRPA1 in normal human dental pulp. (A) As shown for an example, the TRPA1 immunoreactivity, indicated by patches of an electron-dense reaction product (arrow), is localized in the central regions of the axoplasm of myelinated axons in the radicular pulp and (B) adjacent to the axonal membrane of unmyelinated axons in the peripheral pulp. Scale bars = 500 nm.
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Clinical Research Acknowledgments The authors thank Dr Juli Valtschanoff for helpful discussion and careful reading of the manuscript. The authors deny any conflicts of interest related to this study.
References 1. Story GM, Peier AM, Reeve AJ, et al. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 2003;112:819–29. 2. Ji G, Zhou S, Carlton SM. Intact Adelta-fibers up-regulate transient receptor potential A1 and contribute to cold hypersensitivity in neuropathic rats. Neuroscience 2008; 154:1054–66. 3. Karashima Y, Talavera K, Everaerts W, et al. TRPA1 acts as a cold sensor in vitro and in vivo. Proc Natl Acad Sci U S A 2009;106:1273–8. 4. Del Camino D, Murphy S, Heiry M, et al. TRPA1 contributes to cold hypersensitivity. J Neurosci 2010;30:15165–74. 5. Park CK, Kim MS, Fang Z, et al. Functional expression of thermo-transient receptor potential channels in dental primary afferent neurons: implication for tooth pain. J Biol Chem 2006;281:17304–11. 6. Kim HY, Chung G, Jo HJ, et al. Characterization of dental nociceptive neurons. J Dent Res 2011;90:771–6. 7. Kim YS, Son JY, Kim TH, Paik SK, et al. Expression of transient receptor potential ankyrin 1 (TRPA1) in the rat trigeminal sensory afferents and spinal dorsal horn. J Comp Neurol 2010;518:687–98. 8. El Karim IA, Linden GJ, Curtis TM, et al. Human odontoblasts express functional thermosensitive TRP channels: implications for dentin sensitivity. Pain 2011;152:2211–23. 9. Amaya F, Decosterd I, Samad TA, et al. Diversity of expression of the sensory neuron-specific TTX-resistant voltage-gated sodium ion channels SNS and SNS2. Mol Cell Neurosci 2000;15:331–42. 10. Sleeper AA, Cummins TR, Dib-Hajj SD, et al. Changes in expression of two tetrodotoxin-resistant sodium channels and their currents in dorsal root ganglion neurons after sciatic nerve injury but not rhizotomy. J Neurosci 2000;20:7279–89. 11. Bongenhielm U, Nosrat CA, Nosrat I, et al. Expression of sodium channel SNS/PN3 and ankyrin(G) mRNAs in the trigeminal ganglion after inferior alveolar nerve injury in the rat. Exp Neurol 2000;164:384–95. 12. Eriksson J, Jablonski A, Persson AK, et al. Behavioral changes and trigeminal ganglion sodium channel regulation in an orofacial neuropathic pain model. Pain 2005;119:82–94. 13. Akopian AN, Souslova V, England S, et al. The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways. Nat Neurosci 1999;2:541–8. 14. Zimmermann K, Leffler A, Babes A, et al. Sensory neuron sodium channel Nav1.8 is essential for pain at low temperatures. Nature 2007;447:855–8.
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15. Abrahamsen B, Zhao J, Asante CO, et al. The cell and molecular basis of mechanical, cold, and inflammatory pain. Science 2008;321:702–5. 16. Kim YS, Kim YJ, Paik SK, et al. Expression of metabotropic glutamate receptor mGluR5 in human dental pulp. J Endod 2009;35:690–4. 17. Sawada Y, Hosokawa H, Hori A, et al. Cold sensitivity of recombinant TRPA1 channels. Brain Res 2007;1160:39–46. 18. Stucky CL, Dubin AE, Jeske NA, et al. Roles of transient receptor potential channels in pain. Brain Res Rev 2009;60:2–23. 19. Allard B, Couble ML, Magloire H, Bleicher F. Characterization and gene expression of high conductance calcium-activated potassium channels displaying mechanosensitivity in human odontoblasts. J Biol Chem 2000;275:25556–61. 20. Magloire H, Lesage F, Couble ML, et al. Expression and localization of TREK-1 K+ channels in human odontoblasts. J Dent Res 2003;82:542–5. 21. Westenbroek RE, Anderson NL, Byers MR. Altered localization of Cav1.2 (L-type) calcium channels in nerve fibers, Schwann cells, odontoblasts, and fibroblasts of tooth pulp after tooth injury. J Neurosci Res 2004;75:371–83. 22. Okumura R, Shima K, Muramatsu T, et al. The odontoblast as a sensory receptor cell? The expression of TRPV1 (VR-1) channels. Arch Histol Cytol 2005;68: 251–7. 23. Son AR, Yang YM, Hong JH, et al. Odontoblast TRP channels and thermo/mechanical transmission. J Dent Res 2009;88:1014–9. 24. Byers MR, Westenbroek RE. Odontoblasts in developing, mature and ageing rat teeth have multiple phenotypes that variably express all nine voltage-gated sodium channels. Arch Oral Biol 2011;56:1199–220. 25. Davidson RM. Neural form of voltage-dependent sodium current in human cultured dental pulp cells. Arch Oral Biol 1994;39:613–20. 26. Allard B, Magloire H, Couble ML, et al. Voltage-gated sodium channels confer excitability to human odontoblasts: possible role in tooth pain transmission. J Biol Chem 2006;281:29002–10. 27. Atoyan R, Shander D, Botchkareva NV. Non-neuronal expression of transient receptor potential type A1 (TRPA1) in human skin. J Invest Dermatol 2009;129: 2312–5. 28. El Karim IA, Linden GJ, Curtis TM, et al. Human dental pulp fibroblasts express the ‘‘cold-sensing’’ transient receptor potential channels TRPA1 and TRPM8. J Endod 2011;37:473–8. 29. Kochukov MY, McNearney TA, Fu Y, Westlund KN. Thermosensitive TRP ion channels mediate cytosolic calcium response in human synoviocytes. Am J Physiol Cell Physiol 2006;291:C424–32. 30. Beneng K, Renton T, Yilmaz Z, et al. Sodium channel Nav1.7 immunoreactivity in painful human dental pulp and burning mouth syndrome. BMC Neurosci 2010; 11:71. 31. Luo S, Perry GM, Levinson SR, Henry MA. Nav1.7 expression is increased in painful human dental pulp. Mol Pain 2008;4:16. 32. Wells JE, Bingham V, Rowland KC, Hatton J. Expression of Nav1.9 channels in human dental pulp and trigeminal ganglion. J Endod 2007;33:1172–6.
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Expression of Transient Receptor Potential Ankyrin 1 in Human Dental Pulp Yun Sook Kim, PhD,* Hoon Kap Jung, DDS,* Tae Kyung Kwon, DDS,† Chin Soo Kim, DDS, PhD,* Jin Hyun Cho, DDS, PhD,* Dong Kuk Ahn, DDS, PhD,* and Yong Chul Bae, DDS, PhD* Abstract Introduction: Transient receptor potential ankyrin 1 (TRPA1) is activated by noxious cold (<17 C) and contributes to cold and mechanical hypersensitivity after inflammation and nerve injury. Methods: To investigate whether TRPA1 is involved in the mediation of nociception, including noxious cold and cold hypersensitivity in teeth, we examined the expression of TRPA1 and sodium channel Nav1.8 in human dental pulp using fluorescent and electron microscopic immunocytochemistry. Results: TRPA1 was expressed in a large number of axons branching extensively in the peripheral pulp and in a few axons within the nerve bundles in the core of the coronal pulp and in the radicular pulp. Under electron microscopy, TRPA1 immunoreactivity was typically localized near the plasma membrane of unmyelinated axons in the peripheral pulp, suggesting that in these axons it may act as a functional receptor. The proportion of axons expressing TRPA1 in neurofilament 200–positive axons significantly increased in the painful pulp compared with the normal pulp. TRPA1 was also densely expressed in the processes and the cell body of odontoblasts. A large number of axons coexpressed TRPA1 and Nav1.8. Conclusions: These findings support the notion that TRPA1 is involved in the perception of noxious cold and cold hypersensitivity in human dental pulp and that TRPA1-mediated nociception is primarily mediated by axons and odontoblasts in the peripheral pulp. (J Endod 2012;38:1087–1092)
Key Words Cold hypersensitivity, dental pulp, immunohistochemistry, inflammation, Nav1.8, nociception, TRPA1
From the *Department of Oral Anatomy and Neurobiology, School of Dentistry, Kyungpook National University, Daegu, Korea; and †Mir Dental Hospital, Daegu, Korea. Supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2011-0028240). Address requests for reprints to Dr Yong Chul Bae, Department of Oral Anatomy and Neurobiology, School of Dentistry, Kyungpook National University, 188-1, 2-Ga, Samdeok-Dong, Jung-Gu, Daegu 700-412, South Korea. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2012 American Association of Endodontists. doi:10.1016/j.joen.2012.04.024
JOE — Volume 38, Number 8, August 2012
D
ental cold hypersensitivity is one of the most frequently suffered symptoms in many adult dental patients. Yet, our knowledge of the mechanisms of dental cold nociception and cold hypersensitivity are just now beginning to be elucidated. Recently, a family of nonselective cation channels (transient receptor potential [TRP] channels) has been shown to render neurons and other cells that express them on their plasma membrane thermosensitive in a fairly narrow and partially overlapping temperature range. Different TRP channels also mediate the action of a variety of pungent compounds in plant extracts on sensory neurons that is perceived as ‘‘hot’’ (eg, capsaicin via the transient receptor potential vanilloid 1) or ‘‘cold’’ (eg, menthol via the transient receptor potential ankyrin 1 [TRPA1]). TRPA1 is expressed primarily in nociceptive neurons and plays a crucial role in the perception of noxious cold (1). It also mediates the hypersensitivity to cold after inflammation and nerve injury (2–4). Recently, it has been shown that neurons in the rat trigeminal ganglion (TG) that innervate the dental pulp express messenger RNA for TRPA1 and that stimulation with noxious cold (<17 C) causes increased intracellular calcium and evoked currents in these neurons (5, 6), implicating TRPA1 in the mediation of noxious cold within the dental pulp. Large numbers of TRPA1-positive (+) axons also terminate in the dorsal part of the trigeminal sensory nuclei that receive intraoral input (7), suggesting that the oral cavity may be densely innervated by TRPA1+ sensory neurons. In addition, a potential role for TRPA1 in human odontoblasts is now beginning to be elucidated (8). However, evidence for a TRPA1-mediated nociception in the dental pulp is still lacking. Voltage-gated sodium channels (Navs) play a crucial role in the generation and propagation of action potentials in neurons. Among the 9 subtypes of Navs in humans, the tetrodotoxin-resistant Nav1.8 is expressed mainly in small-sized nociceptive neurons in the TG (9, 10) and dorsal root ganglia (DRG) (11, 12). Nav1.8-null mice show a negligible behavioral response to noxious cold (13, 14), and their DRG neurons are also unexcitable in the cold (15), suggesting that Nav1.8 is important for the generation and propagation of action potentials at low temperatures. Recently, it has also been reported that rat TG neurons that innervate the dental pulp express messenger RNA for both TRPA1 and Nav1.8 and respond to temperatures below 17 C (5, 6). However, whether Nav1.8 is involved in the transduction of TRPA1-mediated nociception in human pulp is currently unknown. To address these issues, we examined the expression of TRPA1 and Nav1.8 in human dental pulp using fluorescent and electron microscopic immunohistochemistry.
Materials and Methods Tissue Preparation This study was approved by the Research and Ethics Committee of Kyungpook National University, Daegu, Korea, and informed consent was obtained from all human subjects who participated in the experimental investigation after the nature of the procedure had been fully explained. Twelve pulps from healthy premolars extracted during the course of orthodontic treatment (males, age range: 17–21 years old) and 3 pulps from a painful premolar and molar diagnosed with irreversible pulpitis (males, age range: 18–23 years old) at Kyungpook National University Hospital and Mir Dental Hospital, Daegu, Korea, were used for this study. Four normal pulps were used for immunofluorescent staining for TRPA1 and/or Nav1.8, 5 normal pulps and 3 painful
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Clinical Research pulps for immunofluorescent staining for TRPA1 and/or neurofilament 200 (NF200), and 3 normal pulps for electron microscopic (EM) immunohistochemistry for TRPA1. Immediately after extraction, teeth were stored in 0.1 mol/L phosphate buffer (PB) (pH = 7.4) at 4 C for 1 to 4 hours. Then, the teeth were cut longitudinally with a watercooled high-speed diamond bur, and the pulps were fixed for 2 hours in 4% (w/v) paraformaldehyde (PFA) in 0.1 mol/L PB (pH = 7.4) for immunofluorescence or in a mixture of 4% PFA and 0.05% glutaraldehyde for EM immunohistochemistry. For immunofluorescence, the pulps were cryoprotected in 30% sucrose in PB overnight at 4 C and cut on a freezing microtome at 40 mm. For EM immunohistochemistry, the pulps were cut on a vibratome at 60 mm and also cryoprotected in 30% sucrose in PB overnight at 4 C.
Immunofluorescence and Analysis for Immunopositive Axons For immunofluorescence, sections were permeabilized with 50% ethanol for 30 minutes to enhance penetration, blocked with 10% normal donkey serum (Jackson ImmunoResearch, West Grove, PA) for 30 minutes to mask secondary antibody binding sites, and incubated overnight in a mixture of rabbit anti-TRPA1 antiserum (1:1,000, RA14135; Neuromics, Edina, MN) and mouse anti-Nav1.8 antiserum (1:100, 75-166; NeuroMab, Davis, CA) or in a mixture of rabbit anti-TRPA1 antiserum and mouse anti-NF200 antiserum (1:200,000, N0142; Sigma-Aldrich, St Louis, MO) in phosphate-buffered saline (PBS, 0.01 mol/L, pH = 7.4). After washing with PBS, the sections were incubated with appropriate secondary antibodies (fluorescence isothiocyanate or Cy3-conjugated antibodies raised in donkey, 1:200 in PBS, Jackson ImmunoResearch) for 2 hours. The sections were mounted on slides and examined under a fluorescent microscope (Zeiss Axioplan 2; Carl Zeiss Inc, Jena, Germany) or a laser confocal microscope (LSM 510 META, Carl Zeiss Inc). The quantitative analysis of TRPA1 expression within NF200-immunopositive axons was performed on 3 to 4 sections from each of the 5 healthy pulps and from each of the 3 painful pulps with irreversible pulpitis. Three to 4 images from each section were captured from similar regions in the central and peripheral portions of the coronal pulp with an Exi digital camera (Q-Imaging Inc, Surrey, CA) using 20 objectives (1,360 1,036 pixels) and analyzed using NIH ImageJ software (available at http://rsb.info.nih.gov/ij/). The threshold level for classifying pixels as immunopositive for TRPA1 and NF200 was set between 100 and 120 gray levels from images with 256 gray levels. The area fraction (%) of immunopositive axons (TRPA1/NF200) was presented as the mean the standard error.
Data of the normal pulp and the painful pulp with irreversible pulpitis were compared using an unpaired Student’s t test. Quantitative assessment of colocalization of TRPA1 with Nav1.8 was performed on 3 images from each of the 16 sections of 4 healthy coronal pulps. The omission of the primary or secondary antibodies or preadsorption of the TRPA1 antibody with a blocking peptide at a final concentration of 100 mg/mL completely abolished the specific immunostaining (Fig. 1).
EM Immunohistochemistry The procedure for pre-embedding immunohistochemistry was performed as described elsewhere (7, 16). Briefly, cryoprotected sections were frozen on dry ice for 20 minutes, thawed in PBS, and treated with 1% sodium borohydride in PBS for 30 minutes. To suppress endogenous peroxidase, sections were incubated with 3% H2O2 for 10 minutes and blocked with 10% normal donkey serum for 30 minutes. After overnight incubation with rabbit anti-TRPA1 antiserum (1:1,000, Neuromics), sections were incubated in biotinylated donkey antirabbit antiserum (1:200, Jackson ImmunoResearch) for 2 hours, rinsed, incubated with ExtrAvidin peroxidase (1:5,000, Sigma-Aldrich) for 1 hour, and visualized with nickel-intensified diaminobenzidine. Sections were then osmicated (1% osmium tetroxide in PB), dehydrated in graded alcohols, and embedded in Durcupan ACM (Fluka, Buchs, Switzerland). Thin sections were cut, mounted on formvar-coated single-slot nickel grids, counterstained with Sato’s lead, and examined on a Hitachi H 7500 electron microscope (Hitachi, Tokyo, Japan).
Results Strong immunofluorescent staining for TRPA1 was observed in axons and axon collaterals in human dental pulp (Fig. 2). A small number of axons within nerve bundles wrapped with connective tissue were TRPA1 positive (+) in the radicular pulp and in the core of the coronal pulp (Fig. 2A and B). In contrast, in the peripheral pulp, TRPA1+ axons branched extensively and formed a network in the vicinity of the cell-rich zone corresponding to the plexus of Raschow in the occlusal and the cervical regions (Fig. 2C and D). TRPA1+ axons ascending toward the dentin between odontoblasts were rarely observed. The proportion of NF200+ axons expressing TRPA1 significantly increased in the painful pulp compared with the normal pulp (P < .05). Immunoreactivity for TRPA1 was more intense in the painful pulp than in the normal pulp (Figs. 3 and 4). Immunoreactivity for TRPA1 was also observed in the odontoblasts and their processes (Fig. 5A–D). A large number of TRPA1+ axons were immunostained
Figure 1. Immunofluorescent staining for (A) TRPA1 in normal human dental pulp is completely abolished by (B) preadsorption with a control peptide, proving the specificity of the TRPA1 antiserum (200, scale bars = 50 mm).
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Figure 2. Immunofluorescent staining for TRPA1 in (A) the radicular portion, (B) the core of the coronal portion, and (C and D) the peripheral portion (C, occlusal region; D, cervical region) of normal human dental pulp. (A and B) Relatively sparse TRPA1+ axons in the nerve bundle issue few axon collaterals in the radicular pulp and the core of the coronal pulp. (C and D) TRPA1+ axons branch extensively and form a network in the (C) occlusal and (D) cervical regions of the peripheral pulp (200, scale bars = 50 mm).
for Nav1.8. Of the 450 TRPA1+ axons examined in the coronal pulp in 16 sections from 4 pulps, 177 (39.3%) were Nav1.8+ (Fig. 6A–C). At the electron microscopic level, TRPA1 immunoreactivity was observed in the form of an electron-dense amorphous product in the axoplasm of both myelinated and unmyelinated axons in the radicular pulp and the central portion of the coronal pulp. However, it was usually
Figure 3. The area fraction (%) of TRPA1+ axons in NF200+ nerve fibers in the normal and painful (irreversible pulpitis) pulp samples. *A significant difference between normal and painful pulp samples (P < .05).
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observed near the axolemma of unmyelinated axons in the peripheral pulp (Fig. 7).
Discussion The main findings of this study are that TRPA1 is densely expressed in the axons and odontoblasts in the peripheral portion of human dental pulp and the expression of TRPA1 is increased in the pulpal axons after pulpal inflammation. In addition, approximately 40% of TRPA1+ axons coexpress the sodium channel Nav1.8. TRPA1 is selectively expressed by small- and medium-sized, nociceptive neurons in the DRG and TG and has been implicated in the mediation of noxious cold (1, 7, 17) and the cold and mechanical hypersensitivity after inflammation (18). Recently, Park et al (5) showed that rat TG neurons innervating the dental pulp express messenger RNA for TRPA1 and that the application of icilin or noxious cold to these neurons increases their intracellular calcium and evoked cationic currents, suggesting that TRPA1 may contribute to dental pain evoked by noxious cold. The fact that a large number of pulpal axons in the present study expressed TRPA1 is consistent with a TRPA1-mediated cold nociception within human dental pulp. TRPA1 was densely expressed in a large number of axons that branched extensively outside the connective tissue sheath in the peripheral pulp and in a few axons within nerve bundles in the core of the coronal pulp and in the radicular pulp. Electron microscopy revealed that TRPA1 is expressed mostly in unmyelinated peripheral axons, in which it was typically localized at the axonal membrane where it could act as a functional receptor. In contrast, in myelinated axons in the core of the coronal pulp and in the radicular pulp, TRPA1 was localized in the axoplasm away from the membrane, possibly representing a pool of receptors that is being transported to the peripheral pulp. These findings suggest that the TRPA1-mediated sensitivity to noxious cold is
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Figure 4. Double immunofluorescent staining for (A and D) TRPA1 and (B and E) NF200 in the peripheral region of (A–C) normal human pulps and (D–F) painful pulps. Numbers of axons showing TRPA1 expression in NF200+ axons increase in the painful pulp compared with the normal pulp (200, scale bars = 50 mm).
Figure 5. Immunofluorescent staining for TRPA1 in the (A–C) occlusal and (D) cervical regions of normal human dental pulp. Odontoblasts show intense immunoreactivity for TRPA1 in their cell bodies and in their processes (arrowheads). An arrow in C points to a TRPA1+ axon beneath the odontoblast layer (400, scale bars = 50 mm).
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Figure 6. Double immunofluorescent staining for (A) TRPA1 and (B) Nav1.8 in normal human dental pulp. (C) Many TRPA1+ axons are Nav1.8-positive (arrowheads) (200, scale bar = 20 mm).
mediated primarily by axons in the peripheral pulp rather than in the core of the coronal pulp or in the radicular pulp. In addition, very few axons passing between odontoblasts and entering dentinal tubules were TRPA1 positive, suggesting that axons in the dentinal tubules either express another TRP receptor or are insensitive to cold. In the present study, the proportion of axons expressing TRPA1 within NF200+ axons significantly increased, suggesting that TRPA1 is involved in cold and mechanical allodynia and/or hyperalgesia after dental pulp inflammation. Several previous studies have proposed that odontoblasts may act as sensory cells because they express mechanosensitive K+ channels (19, 20), L-type Ca2+ channels (21), and thermosensitive TRP channels including TRPA1 and the transient receptor potential vanilloid 1 (22, 23). They also express voltage-gated sodium channels (24, 25) and may thus be electrically excitable and able to generate action potentials (26). TRPA1 is functionally expressed in other nonneuronal cells, including keratinocytes (27), fibroblasts (28), and synoviocytes (29), which may act in concert with sensory afferents to monitor the thermal environment. In agreement with 1 recent report (8), in this study we showed that TRPA1 is expressed by odontoblasts, thus providing morphologic evidence that human odontoblasts may be involved in sensing and
transducing noxious cold information. TRPA1 immunoreactivity was strong in the processes and in the cell bodies of the odontoblasts. Because, as described previously, TRPA1+ axons were rarely seen ascending toward the dentinal tubule between odontoblasts, we speculate that noxious cold may be sensed primarily by odontoblast processes rather than by axons in the dentinal tubule. About 40% of TRPA1+ axons coexpressed Nav1.8 in this study, corroborating previous reports that TG neurons that innervate the dental pulp express messenger RNA for both TRPA1 and Nav1.8 (5, 6) and that DRG neurons of Nav1.8-null mice have significantly attenuated the expression of TRPA1 (15). Considering that among the 9 subtypes of Nav channels in humans, Nav1.8 can generate an electrical impulse and is involved in the transmission of nociceptive information under cold conditions and plays a crucial role in pain perception at a low temperature (14), our finding supports the notion that Nav1.8 is involved in the conduction of TRPA1mediated cold nociception in human dental pulp. The TRPA1+ axons in the dental pulp that do not express Nav1.8 may express other Navs, possibly Nav1.7 or Nav1.9, both of which have been shown to be overexpressed after pulpal inflammation and have been implicated in the perception of pain after inflammation and nerve injury (30–32).
Figure 7. Electron micrographs showing immunoperoxidase staining for TRPA1 in normal human dental pulp. (A) As shown for an example, the TRPA1 immunoreactivity, indicated by patches of an electron-dense reaction product (arrow), is localized in the central regions of the axoplasm of myelinated axons in the radicular pulp and (B) adjacent to the axonal membrane of unmyelinated axons in the peripheral pulp. Scale bars = 500 nm.
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Clinical Research Acknowledgments The authors thank Dr Juli Valtschanoff for helpful discussion and careful reading of the manuscript. The authors deny any conflicts of interest related to this study.
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15. Abrahamsen B, Zhao J, Asante CO, et al. The cell and molecular basis of mechanical, cold, and inflammatory pain. Science 2008;321:702–5. 16. Kim YS, Kim YJ, Paik SK, et al. Expression of metabotropic glutamate receptor mGluR5 in human dental pulp. J Endod 2009;35:690–4. 17. Sawada Y, Hosokawa H, Hori A, et al. Cold sensitivity of recombinant TRPA1 channels. Brain Res 2007;1160:39–46. 18. Stucky CL, Dubin AE, Jeske NA, et al. Roles of transient receptor potential channels in pain. Brain Res Rev 2009;60:2–23. 19. Allard B, Couble ML, Magloire H, Bleicher F. Characterization and gene expression of high conductance calcium-activated potassium channels displaying mechanosensitivity in human odontoblasts. J Biol Chem 2000;275:25556–61. 20. Magloire H, Lesage F, Couble ML, et al. Expression and localization of TREK-1 K+ channels in human odontoblasts. J Dent Res 2003;82:542–5. 21. Westenbroek RE, Anderson NL, Byers MR. Altered localization of Cav1.2 (L-type) calcium channels in nerve fibers, Schwann cells, odontoblasts, and fibroblasts of tooth pulp after tooth injury. J Neurosci Res 2004;75:371–83. 22. Okumura R, Shima K, Muramatsu T, et al. The odontoblast as a sensory receptor cell? The expression of TRPV1 (VR-1) channels. Arch Histol Cytol 2005;68: 251–7. 23. Son AR, Yang YM, Hong JH, et al. Odontoblast TRP channels and thermo/mechanical transmission. J Dent Res 2009;88:1014–9. 24. Byers MR, Westenbroek RE. Odontoblasts in developing, mature and ageing rat teeth have multiple phenotypes that variably express all nine voltage-gated sodium channels. Arch Oral Biol 2011;56:1199–220. 25. Davidson RM. Neural form of voltage-dependent sodium current in human cultured dental pulp cells. Arch Oral Biol 1994;39:613–20. 26. Allard B, Magloire H, Couble ML, et al. Voltage-gated sodium channels confer excitability to human odontoblasts: possible role in tooth pain transmission. J Biol Chem 2006;281:29002–10. 27. Atoyan R, Shander D, Botchkareva NV. Non-neuronal expression of transient receptor potential type A1 (TRPA1) in human skin. J Invest Dermatol 2009;129: 2312–5. 28. El Karim IA, Linden GJ, Curtis TM, et al. Human dental pulp fibroblasts express the ‘‘cold-sensing’’ transient receptor potential channels TRPA1 and TRPM8. J Endod 2011;37:473–8. 29. Kochukov MY, McNearney TA, Fu Y, Westlund KN. Thermosensitive TRP ion channels mediate cytosolic calcium response in human synoviocytes. Am J Physiol Cell Physiol 2006;291:C424–32. 30. Beneng K, Renton T, Yilmaz Z, et al. Sodium channel Nav1.7 immunoreactivity in painful human dental pulp and burning mouth syndrome. BMC Neurosci 2010; 11:71. 31. Luo S, Perry GM, Levinson SR, Henry MA. Nav1.7 expression is increased in painful human dental pulp. Mol Pain 2008;4:16. 32. Wells JE, Bingham V, Rowland KC, Hatton J. Expression of Nav1.9 channels in human dental pulp and trigeminal ganglion. J Endod 2007;33:1172–6.
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