- Email: [email protected]
TRPM2 Immunoreactivity Is Increased in Fibroblasts, but not Nerves, of Symptomatic Human Dental Pulp
Basic Research—Biology
TRPM2 Immunoreactivity Is Increased in Fibroblasts, but not Nerves, of Symptomatic Human Dental Pulp Kevin C. Rowland, PhD,*† Courtney B. Kanive, DMD,*† Jason E. Wells, PhD,*† and John F. Hatton, DMD*† Abstract Transient receptor potential (TRP) channels function in diverse processes such as acting as second messenger systems, regulating of ionic concentrations, and aiding in thermoception. TRPM2 channels, members of the melastatin subfamily, mediate calcium influx in response to oxidative stress but during pathological states facilitate hyperexcitability and cellular necrosis via calcium excitotoxicity. We hypothesized that TRPM2 channel expression is upregulated in pulpal tissue of symptomatic teeth with signs of irreversible pulpitis. TRPM2 channel expression was significantly increased in pulp from clinically diagnosed symptomatic teeth compared with pulp from asymptomatic teeth. Additionally, increased TRPM2 expression in symptomatic pulp was the result of increased immunoreactivity in fibroblasts, whereas neural expression of TRPM2 was absent. We provide a possible mechanism explaining the association between TRPM2 channel expression with pain and necrosis. We suggest that TRPM2 channel antagonists could be administered in attempts to inhibit the progression of or even reverse pulpal degradation. (J Endod 2007;33:245–248)
Key Words Fibroblasts, human, immunocytochemistry, pulp, transient receptor potential
From the *Southern Illinois University, School of Dental Medicine, Alton, Illinois; and †Saint Louis University, Center for Advanced Dental Education, St. Louis, Missouri. Address requests for reprints to Dr. Kevin C. Rowland, Section Head of Physiology, Department of Applied Dental Medicine, Southern Illinois University, School of Dental Medicine, Alton, IL 62002. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2007 by the American Association of Endodontists. doi:10.1016/j.joen.2006.11.020
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P
ulpal pathosis evokes many complex processes including excitotoxicity, inflammation, necrosis, and apoptosis (1–3). Excitotoxicity results from an influx of calcium through channels, including nonselective cation channels, and is a prominent mechanism of cell death during an ischemic event (1, 4). Transient receptor potential (TRP) channels and TRP-like channels, including TRPM2 (a melastatin subfamily member), are activated by cellular stress and contribute to ischemia-induced membrane depolarization and intracellular calcium accumulation in neurons (1, 5). Increasing intracellular calcium concentration through TRPM2 can lead to cell death by activating enzymatic degradation, forming free radicals, disrupting mitochondrial functions, and activating the proapoptotic protein ADP-ribose polymerase (ADPR) (2, 6). ADPR increases cationic conductance through TRPM2 channels (7), leading to questions concerning the link between ADPR and Ca2⫹ signaling cell death (8). Because TRPM2 channels possess an intracellular domain activated by ADPR, arachidonic acid metabolites, hydrogen peroxide, tumor necrosis factor ␣, and reactive oxygen species, they likely play a role in sensing the redox status of the cell (9). Also, TRPM2 channels mediate intracellular calcium concentration through a critical component of reactive oxygen species sensitivity (10). Additionally, increased calcium influx, coupled with increased H2O2 and oxygen deprivation, (11) exerts positive feedback enhancing calcium-dependent activation contributing to the irreversibility of the process (12). Furthermore, in the presence of ADPR inhibitors, such as ATP, conductance via TRPM2 channels is decreased (13, 14), preventing massive Ca2⫹ influxes and cell death (1, 5). Because key features of pulpal pathosis, including oxidative stress, increased calcium conductance, and inflammatory mediators are activators of TRPM2, it is plausible that TRPM2 activation plays a role in the progression of pulpal pathosis. However, although inflammation upregulates TRP channel expression of the vanniloid family (TRPV) in the pulp microvasculature of symptomatic teeth (15), no studies have examined a correlation between TRPM2 channel expression in pulpal tissue with pain and necrosis. Therefore, we hypothesized that TRPM2 expression is upregulated in pulp of symptomatic teeth with clinical symptoms of inflammation. Indeed, we show for the first time that increased TRPM2 immunoreactivity is present in pulpal tissue of human teeth presenting with hyperalgesia and allodynia. Furthermore, we show that the increase in TRPM2 is from increased expression in pulpal fibroblasts.
Materials and Methods Sample Collection and Immunocytochemistry All clinical procedures were approved by and conducted according to the rules of the Southern Illinois University and Saint Louis University Internal Review Boards. Human pulpal tissue was obtained from teeth extracted per the patient’s treatment plan in the School of Dental Medicine’s Oral Surgery Department. Teeth from patients who reported pain on thermal (heightened response with delayed recovery) or percussion stimulation (hyperalgesia and allodynia, respectively) were defined as symptomatic. Eight symptomatic teeth were obtained from eight different patients; eight asymptomatic healthy teeth were extracted from eight additional patients. Each symptomatic tooth was blindly paired and immunoreacted with an asymptomatic tooth, forming a total of eight pairs. All teeth included in the study were permanent teeth with fully developed roots and free of visible cracks.
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Basic Research—Biology Because of the lack of a lingering thermal response, 2 of our symptomatic samples were classified as percussion sensitive only. Additionally, because teeth that are only percussion sensitive could be the result of hyperocclusion and not because of irreversible pulpitis, hyperocclused teeth were ruled out before extraction with the use of shimstock. After extraction, teeth were immediately fixed in paraformaldehyde (4% buffered in 0.15 mol/L phosphate-buffered saline [PBS]) at 4°C for at least 24 hours. Pulpal tissue was dissected, cryoprotected overnight in 30% sucrose in PBS at 4°C, frozen in Tissue-Tek O.C.T. compound (Andwin Scientific, Addison, IL), sectioned (12 m) on a cryostat (Vibratome, St. Louis, MO), and collected directly onto Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA). Tissue sections were incubated with 10% normal goat serum in 0.4% Triton X-100 for 1 hour at room temperature and then incubated at room temperature with primary antibodies directed against TRPM2 (1:100; rabbit host, Alomone Labs, Jerusalem, Israel), neurofilament protein (200kD Heavy; chicken host, Abcam, Cambridge, MA: 1:10,000), and, in some experiments, prolyl-4-hydroxylase (1:50; mouse host; GeneTex, San Antonio, TX) overnight in 8% normal goat serum and 0.1% Triton X-100 (Fisher Scientific) in PBS at room temperature. To control for antibody specificity, some sections from each pulp received no primary antibody (which eliminated immunoreactivity) and are referred to as the control samples. Preadsorption control samples could not be produced because TRPM2 antigen is not commercially available. All samples (including control samples) were incubated in the following secondary antibodies: AlexaFluor 555 (goat antirabbit IgG), CY-2 (donkey antichicken), and, in some experiments, AlexaFluor 633 (goat antimouse IgG1) for 1 hour (1:500; Jackson Labs, West Grove, PA).
Image Collection and Statistical Analysis By using appropriate laser lines and filters, stacked optical sections of radicular pulp were collected with an Olympus Fluoview FV300 confocal microscope using a 20⫻ 0.75 NA objective. Olympus Fluoview software (Olympus, Center Valley, PA) was used to calculate the mean fluorescent intensity values within a 10,000 m2 region of the radicular pulp from the experimental (with primary antibody) and control (no primary antibody) images. Signal-to-noise ratios (SNRs) were calculated (experimental fluorescent intensity/control fluorescent intensity), and asymptomatic SNRs were subtracted from symptomatic SNRs for each of the eight paired groups (Table 1). Differences were tested for statistical significance with a Wilcoxon signed rank test (alpha was set at 0.05).
Results We measured fluorescent intensity of TRPM2 immunoreactivity in eight pairs of symptomatic and asymptomatic teeth based on patient-
reported symptoms of thermal and percussion sensitivity (Table 1; see Methods). In 7 of 8 trials, pulp extracted from symptomatic teeth had SNRs (experimental fluorescent intensity/control fluorescent intensity) higher than their matched asymptomatic counterparts, showing that TRPM2 channel immunoreactivity is increased in symptomatic pulps (Fig. 1). Control samples (no primary antibody) had very low levels of immunoreactivity, and example control images are pictured in the insets in Fig. 1. SNR differences between the two groups were statistically significant (Table 1; p ⬍ 0.01, Wilcoxon signed rank test). In a second set of experiments, we aimed to determine the cellular origin of the increased TRPM2 immunoreactivity by incubating the tissue with antibodies specific for axon labeling (antineurofilament) and antibodies used to label fibroblasts by labeling a protein, prolyl-4hydroxylase, which is associated with fibroblast activity. The antibody is specific to the -subunit of prolyl-4-hydroxylase, which reacts with fibroblasts but does not label lymphocytes, monocytes, dendritic cells, and granulocytes (GeneTex, Inc., San Antonio, TX). Representative high-magnification examples of stacked confocal images from asymptomatic and symptomatic pulp are shown in Fig. 2 A–D. Interestingly, TRPM2 channel immunoreactivity was absent in axons from both asymptomatic and symptomatic pulp as revealed by merging the corresponding images (Fig. 2D and D’). Symptomatic pulps had more axonal branches than asymptomatic pulp (compare Fig. 2A with A’), which is consistent with prior studies (16 –18). Fibroblasts, revealed with the fibroblast marker prolyl-4-hydroxylase, constituted the majority of the pulp and had complex branching processes. Fibroblasts of asymptomatc pulp had limited TRPM2 channel immunoreactivity (Fig. 2B and C). In symptomatic pulp, the prolyl-4-hydroxlase reactivity reaction product was concentrated to cell somata (arrows in Fig. 2B’), and fibroblast processes had decreased levels of prolyl-4-hydroxylase activity. TRPM2 channel immunoreactivity was, however, greatly increased in both cell somata and processes of fibroblasts of symptomatic pulp when compared with asymptomatic pulp (compare shades of purple in Fig. 2D and 2D’).
Discussion Overall, the present study shows increased TRPM2 immunoreactivity in pulpal tissue extracted from symptomatic teeth. The increased TRPM2 channel immunoreactivity was localized to cell bodies and processes of fibroblasts and absent from the axons that innervate the pulp. The fibroblasts of symptomatic tissue also exhibited decreased prolyl4-hydroxylase immunoreactivity in their cellular processes. In addition to afferent and efferent axons, the bulk of pulpal tissue contains fibroblasts, odontoblasts, macrophages, endothelial cells, and mast cells (19, 20). Under normal conditions, the aforementioned cell types provide maintenance and structural support and function to re-
TABLE 1. Pulpal and periapical diagnoses, patient reported symptoms, and differences between signal-to-noise ratios Tooth
Pulpal Diagnosis
Periapical Diagnosis
1 2
Irreversible pulpitis Irreversible pulpitis/ partial necrosis Irreversible pulpitis Irreversible pulpitis Irreversible pulpitis Irreversible pulpitis Irreversible pulpitis/ partial necrosis Irreversible pulpitis
Normal Acute apical periodontitis
✓
Acute apical periodontitis Acute apical periodontitis Acute apical periodontitis Acute apical periodontitis Acute apical periodontitis
✓ ✓ ✓ ✓
✓ ✓ ✓
Normal
✓
✓
3 4 5 6 7 8
Hot-sensitive Cold-sensitive
PercussionSNR Differences sensitive (Symptomatic-Asymptomatic)* ✓
⫹3.2 ⫹2.4
✓ ✓ ✓ ✓ ✓
⫹1.5 ⫹8.8 ⫹1.0 ⫺0.8 ⫹11.5 ⫹25.5
(SNR; see Methods) between symptomatic and asymptomatic matched pairs. *p ⬍ 0.01 Wilcoxon Signed-Rank Test.
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Basic Research—Biology
Figure 1. TRPM2 immunoreactivity in symptomatic and asymptomatic human pulp tissue. Low magnification stacked confocal images of radicular pulp samples from asymptomatic teeth (A) and from symptomatic teeth (B). Pulp extracted from symptomatic teeth (B) exhibited increased intensity of TRPM2 channel immunoreactivity compared with pulp extracted from asymptomatic teeth (A). Control samples (no primary antibody) were virtually absent of TRPM2 channel immunoreactivity (insets). Scale bar equals 100 m.
spond to pulpal insults. However, under pathological situations, such as irreversible pulpitis, cells become stimulated to release a variety of molecules that serve as inflammatory mediators, such as bradykinin, prostaglandins, neuropeptides, and interleukins (21–26), leading to the induction of inflammation and sensitization of primary afferent neurons (22). The mechanisms by which nonneural pulpal cells are activated to release these inflammatory mediators are unclear. However, we suggest that because of increased TRPM2 channel expression in fibroblasts, their activation could potentially be a link to sequences that lead to overstimulation of the pulp complex. Our observed increases in TRPM2 channel immunoreactivity leads to the following questions: in the tooth pulp, how are TRPM2 channels activated, and what are the effects of the activation? Mitochondria are possible mechanisms of activation. Free radicals, NAD⫹, and ADPR released from injured mitochondria potentially activate TRPM2 (27), contributing to a vicious cycle culminating in pulpal cell death. There-
fore, the contribution of mitochondria to cellular toxicity could be linked to activation or upregulation of TRPM2 channels. One possible, indirect explanation for our measured association between TRPM2 channel immunoreactivity and a patient’s reported sensation of pain could result from the release of intracellular fibroblast contents onto the axons that innervate the pulp. As fibroblasts become overloaded with Ca2⫹, resulting from increases in TRPM2 channel conductances, they necrose, lyse, and then release intracellular fluid that is typically rich in K⫹, a known activator of neurons, into the extracellular space. High extracellular K⫹, coupled with the increased number of axons innervating symptomatic pulp (16 –18), could explain the strong association. To the best of our knowledge, there are no studies that investigated the concentration of K⫹ in the extracellular fluid of symptomatic teeth. Although TRPM2 channels have been implicated in mediating cell death (1), other members of the TRP family such as TRPV1 have shown
Figure 2. TRPM2 channel immunoreactivity is increased in fibroblasts, but not axons, of symptomatic human pulps. Pulps were reacted with antibodies against an axon marker (neurofilament; A and A’), fibroblast marker (prolyl-4-hydroxylase; B and B’), and TRPM2 (C and C’). D and D’ are merged images of neurofilament, prolyl-4-hydroxylase and TRPM2. Asymptomatic pulps had sparse axon labeling when compared with symptomatic pulps (compare A with A’) and increased prolyl-4-hydroxylase activity in processes of fibroblasts (B vs. B’). Please note the increased TRPM2 channel immunoreactivity in symptomatic pulps (C-C’). Arrows represent somatic localization of prolyl-4-hydroxylase. Scale bar equals 50 m.
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Basic Research—Biology roles in cellular degeneration (28 –30). An upregulation of TRPV1 channel expression in endothelial and smooth muscle cells of symptomatic teeth pulpal vasculature is associated with pain (15). Direct and indirect activation of sympathetic neural fibers inhibits the release of capsaicin-evoked calcitonin gene–related peptide release from pulpal afferent fibers, thereby modulating inflammation and pain (31). In conclusion, we show an increase in TRPM2 channel immunoreactivity in fibroblasts but not axons of painful human pulp tissue. The results from this study have potential clinical benefits in that antagonists such as clotrimazole, flufenamic acid, and econazole (32, 33) target TRPM2 channels and may represent a preoperative therapeutic window to delay the progression of pulpal disease, thereby improving clinical management.
Acknowledgments The authors would like to thank Sandy Sawyer for expert technical assistance. We would like to thank Charles Hildebolt for statistical expertise and reading a prior version of the manuscript. We would also like to thank Jane Gillespie and Allen Otsuka for reading a prior version of the manuscript.
References 1. Aarts MM, Tymianski M. TRPMs and neuronal cell death. Pflugers Arch 2005; 451:243–9. 2. Aarts MM, Tymianski M. TRPM7 and ischemic CNS injury. Neuroscientist 2005; 11:116 –23. 3. Peters E, Lau M. Histopathologic examination to confirm diagnosis of periapical lesions: a review. J Can Dent Assoc 2003;69:598 – 600. 4. Aarts M, Iihara K, Wei WL, et al. A key role for TRPM7 channels in anoxic neuronal death. Cell 2003;115:863–77. 5. Lipski J, Park TI, Li D, et al. Involvement of TRP-like channels in the acute ischemic response of hippocampal CA1 neurons in brain slices. Brain Res 2006;1077:187–99. 6. Lipton P. Ischemic cell death in brain neurons. Physiol Rev 1999;79:1431–568. 7. Perraud AL, Schmitz C, Scharenberg AM. TRPM2 Ca2⫹ permeable cation channels: from gene to biological function. Cell Calcium 2003;33:519 –31. 8. Clapham DE, Runnels LW, Strubing C. The TRP ion channel family. Nat Rev Neurosci 2001;2:387–96. 9. Hara Y, Wakamori M, Ishii M, et al. LTRPC2 Ca2⫹-permeable channel activated by changes in redox status confers susceptibility to cell death. Mol Cell 2002;9:163–73. 10. Tong Q, Zhang W, Conrad K, et al. Regulation of the transient receptor potential channel TRPM2 by the Ca2⫹ sensor calmodulin. J Biol Chem 2006;281:9076 – 85. 11. Schinder AF, Olson EC, Spitzer NC, Montal M. Mitochondrial dysfunction is a primary event in glutamate neurotoxicity. J Neurosci 1996;16:6125–33.
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12. Kuhn FJ, Heiner I, Luckhoff A. TRPM2: a calcium influx pathway regulated by oxidative stress and the novel second messenger ADP-ribose. Pflugers Arch 2005;451: 212–9. 13. Sano Y, Inamura K, Miyake A, et al. Immunocyte Ca2⫹ influx system mediated by LTRPC2. Science 2001;293:1327–30. 14. Harteneck C. Function and pharmacology of TRPM cation channels. Naunyn Schmiedebergs Arch Pharmacol 2005;371:307–14. 15. 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;19:248 – 60. 16. Kimberly CL, Byers MR. Inflammation of rat molar pulp and periodontium causes increased calcitonin gene-related peptide and axonal sprouting. Anat Rec 1988;222:289 –300. 17. Taylor PE, Byers MR. An immunocytochemical study of the morphological reaction of nerves containing calcitonin gene-related peptide to microabscess formation and healing in rat molars. Arch Oral Biol 1990;35:629 –38. 18. Taylor PE, Byers MR, Redd PE. Sprouting of CGRP nerve fibers in response to dentin injury in rat molars. Brain Res 1988;461:371– 6. 19. Cohen S, Burns RC. Pathways of the pulp, 8th ed. St. Louis: Mosby, 2002. 20. Farnoush A. Mast cells in human dental pulp. J Endod 1987;13:362–3. 21. Buck S, Reese K, Hargreaves KM. Pulpal exposure alters neuropeptide levels in inflamed dental pulp and trigeminal ganglia: evaluation of axonal transport. J Endod 1999;25(11):718 –21. 22. Costigan M, Woolf CJ. Pain: molecular mechanisms. J Pain 2000;1(3 Suppl):35– 44. 23. Dray A, Perkins M. Bradykinin and inflammatory pain. Trends Neurosci 1993; 16(3):99 –104. 24. Miyauchi M, Takata T, Ito H, et al. Immunohistochemical demonstration of prostaglandins E2, F2 alpha, and 6-keto-prostaglandin F1 alpha in rat dental pulp with experimentally induced inflammation. J Endod 1996;22:600 –2. 25. Miyauchi M, Takata T, Ogawa I, et al. Immunohistochemical demonstration of prostaglandins in various tissues of the rat. Histochem Cell Biol 1996;105:27–31. 26. Park SH, Hsiao GY, Huang GT. Role of substance P and calcitonin gene-related peptide in the regulation of interleukin-8 and monocyte chemotactic protein-1 expression in human dental pulp. Int Endod J 2004;37:185–92. 27. McNulty S, Fonfria E. The role of TRPM channels in cell death. Pflugers Arch 2005;451:235– 42. 28. Agopyan N, Head J, Yu S, Simon SA. TRPV1 receptors mediate particulate matterinduced apoptosis. Am J Physiol Lung Cell Mol Physiol 2004;286:L563–72. 29. Miyamoto R, Tokuda M, Sakuta T, Nagaoka S, Torii M. Expression and characterization of vanilloid receptor subtype 1 in human dental pulp cell cultures. J Endod 2005;31:652– 8. 30. Veldhuis WB, van der Stelt M, Wadman MW, et al. Neuroprotection by the endogenous cannabinoid anandamide and arvanil against in vivo excitotoxicity in the rat: role of vanilloid receptors and lipoxygenases. J Neurosci 2003;23:4127–33. 31. Hargreaves KM, Bowles WR, Jackson DL. Intrinsic regulation of CGRP release by dental pulp sympathetic fibers. J Dent Res 2003;82:398 – 401. 32. Hill K, Benham CD, McNulty S, Randall AD. Flufenamic acid is a pH-dependent antagonist of TRPM2 channels. Neuropharmacology 2004;47:450 – 60. 33. Hill K, McNulty S, Randall AD. Inhibition of TRPM2 channels by the antifungal agents clotrimazole and econazole. Naunyn Schmiedebergs Arch Pharmacol 2004;370: 227–37.
JOE — Volume 33, Number 3, March 2007
TRPM2 Immunoreactivity Is Increased in Fibroblasts, but not Nerves, of Symptomatic Human Dental Pulp Kevin C. Rowland, PhD,*† Courtney B. Kanive, DMD,*† Jason E. Wells, PhD,*† and John F. Hatton, DMD*† Abstract Transient receptor potential (TRP) channels function in diverse processes such as acting as second messenger systems, regulating of ionic concentrations, and aiding in thermoception. TRPM2 channels, members of the melastatin subfamily, mediate calcium influx in response to oxidative stress but during pathological states facilitate hyperexcitability and cellular necrosis via calcium excitotoxicity. We hypothesized that TRPM2 channel expression is upregulated in pulpal tissue of symptomatic teeth with signs of irreversible pulpitis. TRPM2 channel expression was significantly increased in pulp from clinically diagnosed symptomatic teeth compared with pulp from asymptomatic teeth. Additionally, increased TRPM2 expression in symptomatic pulp was the result of increased immunoreactivity in fibroblasts, whereas neural expression of TRPM2 was absent. We provide a possible mechanism explaining the association between TRPM2 channel expression with pain and necrosis. We suggest that TRPM2 channel antagonists could be administered in attempts to inhibit the progression of or even reverse pulpal degradation. (J Endod 2007;33:245–248)
Key Words Fibroblasts, human, immunocytochemistry, pulp, transient receptor potential
From the *Southern Illinois University, School of Dental Medicine, Alton, Illinois; and †Saint Louis University, Center for Advanced Dental Education, St. Louis, Missouri. Address requests for reprints to Dr. Kevin C. Rowland, Section Head of Physiology, Department of Applied Dental Medicine, Southern Illinois University, School of Dental Medicine, Alton, IL 62002. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright © 2007 by the American Association of Endodontists. doi:10.1016/j.joen.2006.11.020
JOE — Volume 33, Number 3, March 2007
P
ulpal pathosis evokes many complex processes including excitotoxicity, inflammation, necrosis, and apoptosis (1–3). Excitotoxicity results from an influx of calcium through channels, including nonselective cation channels, and is a prominent mechanism of cell death during an ischemic event (1, 4). Transient receptor potential (TRP) channels and TRP-like channels, including TRPM2 (a melastatin subfamily member), are activated by cellular stress and contribute to ischemia-induced membrane depolarization and intracellular calcium accumulation in neurons (1, 5). Increasing intracellular calcium concentration through TRPM2 can lead to cell death by activating enzymatic degradation, forming free radicals, disrupting mitochondrial functions, and activating the proapoptotic protein ADP-ribose polymerase (ADPR) (2, 6). ADPR increases cationic conductance through TRPM2 channels (7), leading to questions concerning the link between ADPR and Ca2⫹ signaling cell death (8). Because TRPM2 channels possess an intracellular domain activated by ADPR, arachidonic acid metabolites, hydrogen peroxide, tumor necrosis factor ␣, and reactive oxygen species, they likely play a role in sensing the redox status of the cell (9). Also, TRPM2 channels mediate intracellular calcium concentration through a critical component of reactive oxygen species sensitivity (10). Additionally, increased calcium influx, coupled with increased H2O2 and oxygen deprivation, (11) exerts positive feedback enhancing calcium-dependent activation contributing to the irreversibility of the process (12). Furthermore, in the presence of ADPR inhibitors, such as ATP, conductance via TRPM2 channels is decreased (13, 14), preventing massive Ca2⫹ influxes and cell death (1, 5). Because key features of pulpal pathosis, including oxidative stress, increased calcium conductance, and inflammatory mediators are activators of TRPM2, it is plausible that TRPM2 activation plays a role in the progression of pulpal pathosis. However, although inflammation upregulates TRP channel expression of the vanniloid family (TRPV) in the pulp microvasculature of symptomatic teeth (15), no studies have examined a correlation between TRPM2 channel expression in pulpal tissue with pain and necrosis. Therefore, we hypothesized that TRPM2 expression is upregulated in pulp of symptomatic teeth with clinical symptoms of inflammation. Indeed, we show for the first time that increased TRPM2 immunoreactivity is present in pulpal tissue of human teeth presenting with hyperalgesia and allodynia. Furthermore, we show that the increase in TRPM2 is from increased expression in pulpal fibroblasts.
Materials and Methods Sample Collection and Immunocytochemistry All clinical procedures were approved by and conducted according to the rules of the Southern Illinois University and Saint Louis University Internal Review Boards. Human pulpal tissue was obtained from teeth extracted per the patient’s treatment plan in the School of Dental Medicine’s Oral Surgery Department. Teeth from patients who reported pain on thermal (heightened response with delayed recovery) or percussion stimulation (hyperalgesia and allodynia, respectively) were defined as symptomatic. Eight symptomatic teeth were obtained from eight different patients; eight asymptomatic healthy teeth were extracted from eight additional patients. Each symptomatic tooth was blindly paired and immunoreacted with an asymptomatic tooth, forming a total of eight pairs. All teeth included in the study were permanent teeth with fully developed roots and free of visible cracks.
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Basic Research—Biology Because of the lack of a lingering thermal response, 2 of our symptomatic samples were classified as percussion sensitive only. Additionally, because teeth that are only percussion sensitive could be the result of hyperocclusion and not because of irreversible pulpitis, hyperocclused teeth were ruled out before extraction with the use of shimstock. After extraction, teeth were immediately fixed in paraformaldehyde (4% buffered in 0.15 mol/L phosphate-buffered saline [PBS]) at 4°C for at least 24 hours. Pulpal tissue was dissected, cryoprotected overnight in 30% sucrose in PBS at 4°C, frozen in Tissue-Tek O.C.T. compound (Andwin Scientific, Addison, IL), sectioned (12 m) on a cryostat (Vibratome, St. Louis, MO), and collected directly onto Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA). Tissue sections were incubated with 10% normal goat serum in 0.4% Triton X-100 for 1 hour at room temperature and then incubated at room temperature with primary antibodies directed against TRPM2 (1:100; rabbit host, Alomone Labs, Jerusalem, Israel), neurofilament protein (200kD Heavy; chicken host, Abcam, Cambridge, MA: 1:10,000), and, in some experiments, prolyl-4-hydroxylase (1:50; mouse host; GeneTex, San Antonio, TX) overnight in 8% normal goat serum and 0.1% Triton X-100 (Fisher Scientific) in PBS at room temperature. To control for antibody specificity, some sections from each pulp received no primary antibody (which eliminated immunoreactivity) and are referred to as the control samples. Preadsorption control samples could not be produced because TRPM2 antigen is not commercially available. All samples (including control samples) were incubated in the following secondary antibodies: AlexaFluor 555 (goat antirabbit IgG), CY-2 (donkey antichicken), and, in some experiments, AlexaFluor 633 (goat antimouse IgG1) for 1 hour (1:500; Jackson Labs, West Grove, PA).
Image Collection and Statistical Analysis By using appropriate laser lines and filters, stacked optical sections of radicular pulp were collected with an Olympus Fluoview FV300 confocal microscope using a 20⫻ 0.75 NA objective. Olympus Fluoview software (Olympus, Center Valley, PA) was used to calculate the mean fluorescent intensity values within a 10,000 m2 region of the radicular pulp from the experimental (with primary antibody) and control (no primary antibody) images. Signal-to-noise ratios (SNRs) were calculated (experimental fluorescent intensity/control fluorescent intensity), and asymptomatic SNRs were subtracted from symptomatic SNRs for each of the eight paired groups (Table 1). Differences were tested for statistical significance with a Wilcoxon signed rank test (alpha was set at 0.05).
Results We measured fluorescent intensity of TRPM2 immunoreactivity in eight pairs of symptomatic and asymptomatic teeth based on patient-
reported symptoms of thermal and percussion sensitivity (Table 1; see Methods). In 7 of 8 trials, pulp extracted from symptomatic teeth had SNRs (experimental fluorescent intensity/control fluorescent intensity) higher than their matched asymptomatic counterparts, showing that TRPM2 channel immunoreactivity is increased in symptomatic pulps (Fig. 1). Control samples (no primary antibody) had very low levels of immunoreactivity, and example control images are pictured in the insets in Fig. 1. SNR differences between the two groups were statistically significant (Table 1; p ⬍ 0.01, Wilcoxon signed rank test). In a second set of experiments, we aimed to determine the cellular origin of the increased TRPM2 immunoreactivity by incubating the tissue with antibodies specific for axon labeling (antineurofilament) and antibodies used to label fibroblasts by labeling a protein, prolyl-4hydroxylase, which is associated with fibroblast activity. The antibody is specific to the -subunit of prolyl-4-hydroxylase, which reacts with fibroblasts but does not label lymphocytes, monocytes, dendritic cells, and granulocytes (GeneTex, Inc., San Antonio, TX). Representative high-magnification examples of stacked confocal images from asymptomatic and symptomatic pulp are shown in Fig. 2 A–D. Interestingly, TRPM2 channel immunoreactivity was absent in axons from both asymptomatic and symptomatic pulp as revealed by merging the corresponding images (Fig. 2D and D’). Symptomatic pulps had more axonal branches than asymptomatic pulp (compare Fig. 2A with A’), which is consistent with prior studies (16 –18). Fibroblasts, revealed with the fibroblast marker prolyl-4-hydroxylase, constituted the majority of the pulp and had complex branching processes. Fibroblasts of asymptomatc pulp had limited TRPM2 channel immunoreactivity (Fig. 2B and C). In symptomatic pulp, the prolyl-4-hydroxlase reactivity reaction product was concentrated to cell somata (arrows in Fig. 2B’), and fibroblast processes had decreased levels of prolyl-4-hydroxylase activity. TRPM2 channel immunoreactivity was, however, greatly increased in both cell somata and processes of fibroblasts of symptomatic pulp when compared with asymptomatic pulp (compare shades of purple in Fig. 2D and 2D’).
Discussion Overall, the present study shows increased TRPM2 immunoreactivity in pulpal tissue extracted from symptomatic teeth. The increased TRPM2 channel immunoreactivity was localized to cell bodies and processes of fibroblasts and absent from the axons that innervate the pulp. The fibroblasts of symptomatic tissue also exhibited decreased prolyl4-hydroxylase immunoreactivity in their cellular processes. In addition to afferent and efferent axons, the bulk of pulpal tissue contains fibroblasts, odontoblasts, macrophages, endothelial cells, and mast cells (19, 20). Under normal conditions, the aforementioned cell types provide maintenance and structural support and function to re-
TABLE 1. Pulpal and periapical diagnoses, patient reported symptoms, and differences between signal-to-noise ratios Tooth
Pulpal Diagnosis
Periapical Diagnosis
1 2
Irreversible pulpitis Irreversible pulpitis/ partial necrosis Irreversible pulpitis Irreversible pulpitis Irreversible pulpitis Irreversible pulpitis Irreversible pulpitis/ partial necrosis Irreversible pulpitis
Normal Acute apical periodontitis
✓
Acute apical periodontitis Acute apical periodontitis Acute apical periodontitis Acute apical periodontitis Acute apical periodontitis
✓ ✓ ✓ ✓
✓ ✓ ✓
Normal
✓
✓
3 4 5 6 7 8
Hot-sensitive Cold-sensitive
PercussionSNR Differences sensitive (Symptomatic-Asymptomatic)* ✓
⫹3.2 ⫹2.4
✓ ✓ ✓ ✓ ✓
⫹1.5 ⫹8.8 ⫹1.0 ⫺0.8 ⫹11.5 ⫹25.5
(SNR; see Methods) between symptomatic and asymptomatic matched pairs. *p ⬍ 0.01 Wilcoxon Signed-Rank Test.
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Figure 1. TRPM2 immunoreactivity in symptomatic and asymptomatic human pulp tissue. Low magnification stacked confocal images of radicular pulp samples from asymptomatic teeth (A) and from symptomatic teeth (B). Pulp extracted from symptomatic teeth (B) exhibited increased intensity of TRPM2 channel immunoreactivity compared with pulp extracted from asymptomatic teeth (A). Control samples (no primary antibody) were virtually absent of TRPM2 channel immunoreactivity (insets). Scale bar equals 100 m.
spond to pulpal insults. However, under pathological situations, such as irreversible pulpitis, cells become stimulated to release a variety of molecules that serve as inflammatory mediators, such as bradykinin, prostaglandins, neuropeptides, and interleukins (21–26), leading to the induction of inflammation and sensitization of primary afferent neurons (22). The mechanisms by which nonneural pulpal cells are activated to release these inflammatory mediators are unclear. However, we suggest that because of increased TRPM2 channel expression in fibroblasts, their activation could potentially be a link to sequences that lead to overstimulation of the pulp complex. Our observed increases in TRPM2 channel immunoreactivity leads to the following questions: in the tooth pulp, how are TRPM2 channels activated, and what are the effects of the activation? Mitochondria are possible mechanisms of activation. Free radicals, NAD⫹, and ADPR released from injured mitochondria potentially activate TRPM2 (27), contributing to a vicious cycle culminating in pulpal cell death. There-
fore, the contribution of mitochondria to cellular toxicity could be linked to activation or upregulation of TRPM2 channels. One possible, indirect explanation for our measured association between TRPM2 channel immunoreactivity and a patient’s reported sensation of pain could result from the release of intracellular fibroblast contents onto the axons that innervate the pulp. As fibroblasts become overloaded with Ca2⫹, resulting from increases in TRPM2 channel conductances, they necrose, lyse, and then release intracellular fluid that is typically rich in K⫹, a known activator of neurons, into the extracellular space. High extracellular K⫹, coupled with the increased number of axons innervating symptomatic pulp (16 –18), could explain the strong association. To the best of our knowledge, there are no studies that investigated the concentration of K⫹ in the extracellular fluid of symptomatic teeth. Although TRPM2 channels have been implicated in mediating cell death (1), other members of the TRP family such as TRPV1 have shown
Figure 2. TRPM2 channel immunoreactivity is increased in fibroblasts, but not axons, of symptomatic human pulps. Pulps were reacted with antibodies against an axon marker (neurofilament; A and A’), fibroblast marker (prolyl-4-hydroxylase; B and B’), and TRPM2 (C and C’). D and D’ are merged images of neurofilament, prolyl-4-hydroxylase and TRPM2. Asymptomatic pulps had sparse axon labeling when compared with symptomatic pulps (compare A with A’) and increased prolyl-4-hydroxylase activity in processes of fibroblasts (B vs. B’). Please note the increased TRPM2 channel immunoreactivity in symptomatic pulps (C-C’). Arrows represent somatic localization of prolyl-4-hydroxylase. Scale bar equals 50 m.
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Basic Research—Biology roles in cellular degeneration (28 –30). An upregulation of TRPV1 channel expression in endothelial and smooth muscle cells of symptomatic teeth pulpal vasculature is associated with pain (15). Direct and indirect activation of sympathetic neural fibers inhibits the release of capsaicin-evoked calcitonin gene–related peptide release from pulpal afferent fibers, thereby modulating inflammation and pain (31). In conclusion, we show an increase in TRPM2 channel immunoreactivity in fibroblasts but not axons of painful human pulp tissue. The results from this study have potential clinical benefits in that antagonists such as clotrimazole, flufenamic acid, and econazole (32, 33) target TRPM2 channels and may represent a preoperative therapeutic window to delay the progression of pulpal disease, thereby improving clinical management.
Acknowledgments The authors would like to thank Sandy Sawyer for expert technical assistance. We would like to thank Charles Hildebolt for statistical expertise and reading a prior version of the manuscript. We would also like to thank Jane Gillespie and Allen Otsuka for reading a prior version of the manuscript.
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