0099-2399/90/1602-0078/$02.00/0 JOURNAL OF ENDODON]ICS Copyright 9 1990 by The American Association of Endodontists
Printed in U.S.A.
VOL. 16, NO. 2, FEBRUARY 1990
Effects of Injury and Inflammation on Pulpal and Periapical Nerves Margaret R. Byers, PhD, Patrick E. Taylor, DDS, MSD, Bertrand G. Khayat, DDS, MSD, and Charles L. Kimberly, DDS, MSD
Several studies dealing with the reactions of dental nerve fibers to injury and inflammation are reviewed in this article. The subgroup of dental nerve fibers that contains calcitonin gene-related peptide (CGRP) was examined by immunocytochemistry at various times (1 to 35 days) after one of three degrees of injury: (a) Mild: Four days after making shallow cavities into cervical dentin of first molars of anesthetized adult rats, we found that CGRP fibers had sprouted into the subjacent odontoblast layer and dentin, and then returned to normal by 3 wk. (b) Intermediate: If the cervical cavities were acid etched, we found damage to the odontoblast layer, microabscess formation, and sprouting of CGRP fibers near the abscess, with subsequent formation of reparative dentin and healing. (c) Severe: If the pulp was exposed, a variety of reactions could occur, the most prevalent of which was a severe necrosis leading to development of periapical lesions. Analysis of the progressive stages of pulpal abscess and necrosis showed sprouting CGRP nerve fibers (a) at the retreating interface between abscess and vital pulp; (b) in periapical areas during onset of lesions; and (c) around chronic abscesses in granulomatous periodontal tissues. These studies are discussed in relation to various dental clinical problems such as hypersensitive teeth, episodic toothache, early onset of periapical lesions, dental anesthesia, and possible roles for sensory fibers and neuropeptides in tissue defense and healing.
Following mandibular nerve injury extensive reinnervation of sensory fibers to pulp and dentin occurs (2). A small proportion of dental nerve fibers are sympathetic as demonstrated by their cytochemistry (norepinephrine, neuropeptide Y) and by their loss following sympathetic ganglionectomy (4, 5). The sympathetic fibers primarily end on blood vessels deep in pulp. Another group of nerves which contain vasoactive intestinal peptide are more numerous than the sympathetic axons and may be parasympathetic, because they are not affected by trigeminal lesions or sympathectomy (4). The innervation of teeth is therefore complex and has diverse sensory functions, autonomic functions, and cytochemistry. During the past few years, the subgroup of dental sensory fibers that contains calcitonin gene-related peptide (CGRP), substance, P and neurokinin A has been the subject of much research (6, 7); these fibers form numerous endings in coronal pulp and dentin, along many pulpal blood vessels, and in periodontal tissue. Studies in other tissues show that CGRP and substance P are released by sensory fibers and exert profound effects that alter blood flow, inflammatory and immune responses, and connective tissue cells (8.9). Several questions can be asked about these sensory nerve fibers: What are their functions in pulpal and dentinal biology? Do they interact synergistically (or antagonistically) with nonpeptidergic sensory fibers or with autonomic fibers? What are their sensory, properties? How do they respond to injury and inflammation? What is their contribution to clinical problems such as hypersensitive dentin, toothache, and difficult anesthesia of inflamed teeth? We have recently analyzed the morphological changes of these sensory fibers to specific types of injury using immunocytochemistry for CGRP (10-13). Our previous autoradiographic studies of experimental cavities in rat molars (14) confirmed the earlier electron microscopic studies of Lilja et al. (15) demonstrating aspiration of nerve fibers into dentin under acid-etched, desiccated cavities; the effects on underlying pulp and nerve fibers were proportional to the depth of cavity and the degree of etching and air drying. From that work we devised the following injury models in rat molars: (a) Shallow cervical cavity (unetched)--This affects the sparsely innervated cervical root pulp without destroying the odontoblast layer (10). (b) Deeper cervical cavity with etching--This causes some odontoblast damage and microabscess formation followed by production of reparative dentin and healing (I 3). (c) Coronal pulp exposure leading to pulpal necrosis--Tooth reactions to pulp exposure included temporary pulp polyp formation, progressive liquefying or coagulation necrosis, and early onset of periapical lesions ( 11, 12).
The distribution of nerve fibers in tecth has been extensively documented during the past 30 yr using histological, electron microscopic, axonal transport, and immunocytochemical methods (1-3). Most of the nerve fibers pass through the roots, then make numerous branches in the coronal pulp, and terminate in the peripheral pulp or in the inner 0.1 mm of dentin. The sensory nerve fibers, which comprise the great majority of dental innervation, originate in the trigeminal ganglion and range in size from C fibers (0.1 to 1.0 um in diameter) to A-/~ (l to 5 ~m) and A-/3 fibers (6 to 10 ~m). 78
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In this article, we will review our studies concerning CGRP nerve reactions to these three types of injury. Although the CGRP (substance P, neurokinin A) nerve fibers are only one component of dental innervation, they play a principal role in neurogenic inflammation and are thus very important for pulpal and periapical pathological reactions and dental pain (16).
consent) by CGRP immunocytochemistry. The staining was most successful in teeth that were cleaved longitudinally immediately after extraction for optimal access of fixative to the pulp tissue. The immunocytochemistry procedures were similar to those used for rats (10-13), and both antirat and antihuman CGRP antibodies (Cambridge Research Biochemicals) worked well.
M A T E R I A L S AND M E T I I O D S
RESULTS
Full details of the immunocytochemical methods are given in Taylor et al. (10), Kimberly and Byers (I l), Khayat et al. (12), and Taylor and Byers (13). For all rat surgeries, the animals were deeply anesthetized with intraperitoneal Equithesin (4.25% chloral hydrate, 0.97% sodium pentobarbital) at 0.3 ml/100 g body wt. Their recovery from anesthesia was rapid and uneventful. During the postoperative period, the rats were weighed and examined daily and were found to quickly regain normal grooming, feeding, and weight gain and to exhibit an overall healthy appearance. Postinjury survival times ranged from 1 to 35 days, after which the rats were deeply anesthetized with 2.5% sodium pentobarbital (i.p.) and perfusion fixed with a mixturc of formaldehyde (4%) and picric acid (0.2%) in 0.1 M phosphate buffer. Standard immunocytochemical procedures were used as described earlier (10-13), the specificity of staining of nerve fibers was confirmed by standard controls, including omission of key reagents or preadsorption of the primary antibody with synthetic CGRP. The distribution of CGRP immunoreactive (IR) nerve fibers was analyzed in serial sections from injured and contralateral normal teeth using a Zeiss light microscope, and in some cases quantitative measurements were made. The methods for autoradiographic mapping of trigeminal dental sensory fibers (Figs. 1 and 2) are reviewed elsewhere (2). Some normal human teeth that had been extracted for orthodontic purposes were also analyzed (with the patients'
..
:~ - ~
~.=;:;:~,., ~:~" ,~.~.
FIGs 1 and 2. Autoradiographs showing the location of trigeminal sensory nerve fibers in rat molars. Most terminal branching was in the crown, with many fibers entering 0.1 mm into regular dentin (arrows), but avoiding reparative dentin (RD). Below the cervical line (dashes) very few fibers entered dentin (original magnification: Fig. 1, x87; Fig. 2, x345). Scale bars: 0.1 mm. Figure 2 is reprinted from Byers et al. (14) with permission.
Uninjured M o l a r s
The autoradiographic mapping that shows all trigeminal dental fiber types (Figs. 1 and 2) can be compared with the subgroup of CGRP-IR fibers in immunocytochemical sections of normal adult rat molars (Figs. 3 and 4). In both cases, the labeled nerve fiber bundles in the root pulp branched extensively into the coronal pulp forming the peripheral plexus of Raschkow, innervating the odontoblast layer, and entering the dentin to a distance of up to 0. I ram. There was sparse innervation of reparative dentin, and greatly reduced inncrvation in dentinal tubules of the roots, lnterradicular dentin was not innervated in normal rat molars.
Mild Injury
Four days after making shallow cavities into the cervical dentin of rat first molars, numerous CGRP-IR sprouts had formed in the underlying odontoblast layer (Figs. 5 and 6). Quantitative studies showed that the innervation from the pulp tip along the anterior mid-line pulpal surface had increased an average of more than 0.6 mm to extend into cervical root areas that normally were not innervated. Electron microscopy showed that these were new CGRP-IR nerve terminals and not preexisting nerve fibers with altered cytochemistry. The sprouting reaction had partially subsided in teeth examined 6 to 11) days postinjury and was gone by 21 days, as described by Taylor et al. (10).
FIGS 3 and 4. Immunocytochemical sections showing CGRP-IR nerve fibers branching extensively in the coronal pulp and entering into dentin for up to 0.1 mm, but avoiding reparative dentin (RD). Dashes show the cervical line (original magnification: Fig. 3, x50; Fig. 4, x130). Scale bars: 0.1 ram. Figure 4 is reprinted from Kimberly and Byers (11) with permission.
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FIGS 5 and 6. Immunocytochemical sections of a matched pair of first maxillary rat molars. In the contralateral control (Fig. 5), CGRPIR fibers were numerous near the pulp horn tip and extended along the pulp dentin border to the cervical line (dashes). The injured tooth (Fig. 6) was drilled at the cervical region 4 days earlier; numerous CGRP-IR nerve fibers have sprouted below the injury and have extended into previously sparsely innervated cervical regions along the pulp-dentin border (original magnification x80). Scale: 0.1 mm. Reprinted from Taylor et al. (10) with permission.
Microabscess Formation and Repair If the cervical cavities in rat molars were deep or were etched for 10 to 30 s with 37% phosphoric acid, microabscesses were found in the adjacent pulp 4 days later (Fig. 7) with progressive reparative dentin formation at 7 to 35 days (Figs. 8 to 10). In many cases, the injured sites were completely covered by reparative dentin by 35 days. The CGRP-IR fibers formed numerous branches in the pulp adjacent to the microabscesses. They were intermingled with the odontoblasts that were forming reparative dentin, and their sprouting subsided once the abscessed pulp had been replaced by reparative dentin, as described in Taylor and Byers (13).
Pulpal Necrosis
FIG 7. Four days after inducing the formation of an abscess (,) in cervical pulp, numerous terminal branches of CGRP-IR nerve fibers were found adjacent to the injured tissue or invading the lesion (original magnification x170). Scale: 0.1 mm
after pulpal exposure, even though much of the pulp tissue was still vital (12). In the 35-day postinjury group, when pulp tissue had been completely consumed by the advancing pulpitis, abscesses extended into the periodontal ligament and alveolar bone (Figs. 14 and 15). The CGRP-IR fibers continued to sprout around those periodontal abscesses and appeared to be actively attracted to those sites; cytochemical changes had also occurrcd by 35 days postinjury in the nearby alveolar nerves, with many more fibers staining for CGRP (ll).
Although we attempted to make pulpal exposures the same way in all cases, several reactions were found in the teeth with exposed pulp. In some, pulpal tissue was not severely damaged and had begun to form pulp polyps by 5 to 6 days postinjury (Fig. I 1); in others, a coagulation necrosis had replaced the coronal pulp by that time (Fig. 12); and in a third group liquefying necrosis was in progress (Fig. 13). Characteristic changes in the CGRP-1R nerves were found for all three reactions, all of which included sprouting of CGRP-1R fibers along the border and clustering of axons in the core of the surviving pulp. for further details see Kimberly and Byers (11) and Khayat et al. (12).
Extracted human teeth were analyzed by CGRP immunocytochemistry. In immature and mature permanent premolar or molar teeth numerous CGRP fibers were found, with many branches in the subodontoblast region, odontoblast layer, and predcntin and dentin of cervical (Fig. 16) and coronal areas.
Periapical Lesions
DISCUSSION
Quantitative measurements of periapical tissue showed that periapical lesions were beginning to develop by 5 to 8 days
The interactions between nerve fibers, pulp cells, immune cells, vasculature, and pulpal fluid are complex and dynamic.
Normal Human Teeth
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Fles 8 to 10. These teeth all received a cervical cavity that was then acid etched. An abscess (*) can be seen 7 days' postinjury (Fig. 8) with numerous CGRP-IR fibers in adjacent pulp. By 11 days postinjury (Fig. 9), large blocks of reparative dentin were forming with numerous CGRPIR fibers in the adjacent pulp. By 35 days (Fig. 10), reparative dentin had completely sealed off the injury site, and CGRP-IR fibers were less numerous and less intensely stained for CGRP-IR (original magnification x65). Scale: 0.1 mm.
FIGs 11 to 13. Three teeth showing the varied responses at 5 to 6 days after exposure of rat molar pulp, incJuding pulp polyp formation (Fig. 11), coagulation necrosis (Fig. 12), and liquefying necrosis (Fig. 13) (original magnification x80). Scale: 0.1 mm
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FIGS 14 and 15. The difference between CGGP-IR fibers in a normal rat molar periapical region (Fig. 14) and at 35 days after initiating irreversible pulpitis (Fig. 15) is shown. The abscess had extended out of the pulp into an abnormally widened periapical socket and numerous CGRP-IR sprouts were attracted toward the abscess (,). In normal tissue only a few nerves stained for CGRP-IR are seen, compared with the numerous CGRP-IR axons near abscessed ligament (original magnification x45). Scale: 0.1 mm. Reprinted from Kimberly and Byers (11 ) with permission.
NERVES SENSORY (non-PeDtide) (peptides)
SYMPATHETIC & ParasymD
>ULP CELLS ..Odontoblasts 9Fibroblast s Immune Cells
FLUID DYNAMICS PulDal Oentinal Vascular
"Three Worlds* M.C.Escher (1955)
F~G 16. Human permanent left maxillary first premolar with numerous CGRP-IR nerve fibers in the subodontoblast region, in the odontoblast layer (=), and in dentin (D) (original magnification x190). Scale: 0.1 mm.
FIG 17. M. C. Escher's image "Three Worlds" (1955) has been used here to illustrate the point that various dental nerve fibers, pulp cells, and factors affecting fluid dynamics in teeth interact in numerous complex ways that can activate pain sensations (represented by the fish). It is a challenge for dental research to define those interactions and to devise optimal preventive measures and pain management techniques. Repnnted with permission from M. C. Escher Heirs, 9 1988 M. C. Escher Heirs/Cordon Art-Baarn, Holland.
Vol. 16, No. 2, February 1990
Clearly, some combinations of those interactions cause pain and others do not. Although there is still much to learn about neural and cellular interactions in pulp patholosis, several general statements can be made based on clinical observations (I 7), on numerous experiments by Br~instr6m (18) and others, and on our series of immunocytochemical experiments in rats.
Hypersensitive Dentin and Toothache Many experiments (e.g. 18-20) have suggested that fluid movement in dentinal tubules that are open to the oral environment can cause dental hypersensitivity by stimulating nerve fibers along the pulp-dentin border. Our recent experiments and those by Plackova (21) suggest that sprouting of dental nerve fibers may also have occurred; if so, then the hydrodynamic stimuli to exposed dentin would activate many more nerve fibers than normal. The neural morphological changes were dynamic, with rapid increases in terminal branching and reversion to normal over a period of days. Such temporary fluctuations in neural density may affect the duration of tooth hypersensitivity, as can progressive occlusion of the exposed dentinal tubules (22). Similarly, episodic toothache may include neural fluctuations, with cycles of increased nerve fibers and peptide cytochemical alteration followed by decreases, perhaps a~ociated with cycles of abscess expansion and pulpal healing. Recent immunocytochemical studies of human teeth suggest that there may be sprouting of dental nerves and altered neuropeptides associated with painful caries (23), but more immunocytochemical work needs to be done concerning histopathological correlations with clinical pain.
Dental Anesthesia A regional nerve block frequently fails to affect an inflamed tooth even though adjacent healthy teeth are well anesthetized (24). Our observation that neuropeptides such as CGRP were elevated in trunk axons of trigeminal nerves that innervated inflamed tissue (11~ suggests that those fibers may have an altered capacity for anesthesia due to cytochemical changes extending throughout the affected nerve fibers. That suggestion is supported by recent studies showing rapid alterations in neuropeptide mRNAs in dorsal root ganglia and spinal cord neurons within hours of inducing inflammation (25). Since immunocytochemistry shows relatively large chemical changes for trigeminal axons, one could certainly postulate subtle changes in membrane molecular structure that would affect anesthesia sensitivity; but further work is needed to determine whether such changes occur.
Periapical Lesions Our studies ofperiapical lesions found significant widening of the socket and sprouting of CGRP-IR periapical fibers when vital pulp was still present (12). This suggested that neuropeptides and sensory nerve fiber branching may have contributed to lesion development. Other etiologic factors such as diffusion of bacterial products from the pulpal abscesses may also have contributed. Involvement of a neurogenie mechanism in the onset of periapical lesions is sup-
Dental Nerves and Inflammation
83
ported by evidence for stimulation of blood flow, inflammatory reactions, immune cell activity, and other cellular functions by neuropeptides (8, 9, 26). Much still remains to be determined concerning the role of neuropeptides and neural reactions in tissue defense and healing. Although the inflammatory actions of some sensory nerve fibers are well recognized (26), we should perhaps consider those nerve fibers to be activated following injury primarily for facilitation of inflammatory reactions and tissue healing, with sensory activity as a secondary function. Recent studies of other dental injury models such as orthodontic displacement in rats (27) give further evidence of injuryinduced changes in neural structure, suggesting that such changes may be common to most (or all) dental injuries. The evidence for CGRP innervation of human teeth (Fig. 16) suggests that information gained from animal experiments will have general relevance for human dental biology and pathology. Evidence for interactions between peptidergic sensory fibers and sympathetic fibers in pathologic reactions comes from studies of experimental arthritis (25, 26) or of injured optic tissues (28) in which neural sprouting or neuropeptide changes were associated with both sensory and sympathetic nerve reactions. A full understanding of neural reactions to dental injury and inflammation should therefore include studies of sympathetic, parasympathetic, and nonpeptidergic sensory fibers as well as of the peptidergic sensory fibers and their interactions with pulp (Fig. 17). Finally, evidence for complex interactions between macrophages, leukocytes, and neuropeptides (9) includes stimulation of nerve growth factor production (29), suggesting that nerve growth factor may be a critical factor in controlling the nerve sprouting reactions to dental injury and inflammation (30). This research was supported by NIH Grant OE 05159 to Dr. Byers end by NIH Grant 2-S07-RR05346 (BSRG) and the Graduate Endodontics Fund, University of Washington. Drs. C. L. Kimbedy and P. E. Taylor received salary support from the U.S. Navy. Dr. B. G. Khayat received partial salary support from NIH Grant AG 00122. We thank Dr. G. Harrington, Dr. R. Oswald, and Mr. P. E. Redd for helpful discussions. We also thank Kelly B MecJfl for exten,wve expert technical assistance, Diane Ryba for photographic work, and Nancy Sutton for secretarial work. Our experiments conformed to guidelines of the Human Subjects and Animal Care Committees of the University of Washington. Dr. Byers is a research professor. Departments of Anesthesiology, Endodont~s, and Biologm_,alStructure, University of Washington, Seattle, WA. Drs. Khayat, Kimberly, and Taylor are affiliated with the Department of Endodontics, University of Washington. Address requests for reprints to Dr. Byers, Department of Anesthesiolngy, RN-10, University of Washington, Seattle, WA 98195.
References 1. Gunli T. Morphological work on the sensitivity of dentin. Arch Histol Japn 1982;45:45-60. 2. Byers MR. Dental sensory receptors. Int Rev Neurobio11984;25:39-93. 3. Maeda T, Iwanaga T, Fulita T. Kobayanashi S. Immunohcstochemical demonstration of nerves in the predentin and dentin of human third molars with the usa of an antiserum against neurofilament protein (NFP). Cell Tissue Res 1986;243:469-75 4. Akai M, Wakisaka S. The dlstribuhon of peptidergic nerves. In: Inoki R, Kudo T, Olgart L, eds. Dynamic aspects of the dental pulp. London: Chapman and Hall Ltd. (in press). 5. Edwall B, Gazelius B, Frazekas A, Theodorsson-No~eim E, Lundberg J M Neuropeptide Y (NPY) and sympathetic control of blood flow in oral mucosa and dental pulp in the cat. Acta Physiol Scand 1985:125:253-64. 6. Gazelius B, Edwall B, Olgart L, Lundberg JM, Hokfelt T, Fisher J A Vasodilatory effects and coexistence of calcitonin gene-related peptide (CGRP) and substance P in sensory neurons of cat dental pulp. Acta Physiol Scand 1987;130:33-40.
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7. Silverman JD, Kruger L. Calcitonin-gene-retated-peptide immunoreact}ve innervation of the rat head with empelasis on specialized sensory structure. J Comp Neurol 1989;280:303-30. 8. Brain SD, Williams TJ, Tippins JR, Morris HR, Maclntyre I. Calcitonin gene-ralated peptide is a potent vasodilator. Nature 1985;313:54-6. 9. Payan DG, McGillis JP, Renold FK, Mitsuhashi M, Goet.zl EJ. The role of brain peptides in neuroimmunomodulation. Ann NY Acad Sci 1987;496:18291. 10. 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. 11. Kimberly CL, Byers MR. Inflammation of rat molar pulp and penodontium causes increased calcitonin gene related pepede and axonal sprouting. Anat Rec 1988;222:289-300. 12. Khayat BG, Byers MR, Taylor PE, Mecifi KB, Kimberly CL Responses of nerve fibers to pulpal inflammation and ponapical lesions in rat molars demonstrated by calcitonin gene-related peptide immunocytochemistry. J Endodon 1988;14:577-87. 13. Taylor PE, Byers MR. Response of CGRP-immunoreactive nerve fibers to limited pulpitis and healing in rat molars. Arch Oral Biol (in press). 14. Byers MR, Narhi MVO, Mecifi KB. Acute and chronic reactions of dental sensory nerve fibers to cavities and desiccation in rat molars. Anat Rec 1988;221:872-83. 15. IJIja J, Nordenvall KJ, Brannstrom M. Dentin sensitivity, odontoblasts and nerves under desiccated or infected experimental cavities. Swed Dent J 1982;6:93-103. 16. Olgart LM. The role of local factors in dentin and pulp intradental pain mechanisms. J Dent Res 1985;64(spe~al issue):572-8 17. Trowt)ridge HO. Intradental sensory units: physK)k~jlcal and clinKT,al aspects. J Endodon 1985;11:489-98. 18. Brannstr0m M. Dentin and pulp in restorative dentistry. London: Wolf Medical Publications, 1981.
Joumal of Endodontics 19. Karlsson UL, Penney DA. Natural desensitization of exposed tooth roots in dogs J Dent Res 1975:54:982-6. 20. Hirvonen TJ, Narhi MVO, Hakumaki MDK. The excital~lity of dog ;~JIp nerves in relation to the condition of dentin surface. J Endodon 1984;10: 294-8. 21. Plackova A. Pathologic changes in the innervation of the dental pulp during carious process. J Dent Res 1966;45:62-5. 22. Pashley D. Dentin-predentin complex and its permeability: physiologic overview. J Dent Res 1985;64(speoal issue):613-20. 23. Yamaura Y. Immunohistochemc, al investigations on the nerve fibers of the dental pulp after the cavity preparation. Jpo J Conserv Dent 1987;30:82438. 24. Brown RD. The failure of IocaJ anesthesia in acute inflammation. Br Dent J 1981;151:47-51. 25. ladarola MJ, Draisoi G. Elevation of spinal cord dynorp~in mRNA compared to dorsal root ganglio~ beptide mRNAs during peripheral inflammation. In: Bessen JM. Guilbaud G, eds. The arthritic rat as a model of clinkcal pare? New York: Elsevier, 1988;173-83. 26. Levine JD, Coderre T J, Basbaum AI. The peripheral nervous system and the inflammatory process. Pain Res Clin Managem 1988:3:33-43. 27. Kvinnsland I, Kvinnsland S. Changes in CGRP-immunoreact/ve nerve fibers during experimental tooth movement in rats. Eur J O ~ (in press). 28. Tererlghi G, Zhen(j SQ, Unger WG, Polak J. Morphological changes of sensory CGRP-immunoreactive and sympathetr nerves in peripheral tissues following chronic denervation. Histochemistry 1986;86:89-95. 29. IJndhotm D, Heumann R, Meyer M, Thoenen H. Intedeukin-1 regulates synthesis of nerve growth factor in non-neuronal cells of rat sciatic nerve. Nature 1987;330:658-9. 30. Byers MR, Bothwell MA, Mecifi KB, Schatteman GC. InteractK)ns of nerves and pulp cells in normal and injured teeth analysed by LM and EM immunocytochemistry for NGF-receptor. Neurosci Abstracts 1988;14:1169.
Printed in U.S.A.
VOL. 16, NO. 2, FEBRUARY 1990
Effects of Injury and Inflammation on Pulpal and Periapical Nerves Margaret R. Byers, PhD, Patrick E. Taylor, DDS, MSD, Bertrand G. Khayat, DDS, MSD, and Charles L. Kimberly, DDS, MSD
Several studies dealing with the reactions of dental nerve fibers to injury and inflammation are reviewed in this article. The subgroup of dental nerve fibers that contains calcitonin gene-related peptide (CGRP) was examined by immunocytochemistry at various times (1 to 35 days) after one of three degrees of injury: (a) Mild: Four days after making shallow cavities into cervical dentin of first molars of anesthetized adult rats, we found that CGRP fibers had sprouted into the subjacent odontoblast layer and dentin, and then returned to normal by 3 wk. (b) Intermediate: If the cervical cavities were acid etched, we found damage to the odontoblast layer, microabscess formation, and sprouting of CGRP fibers near the abscess, with subsequent formation of reparative dentin and healing. (c) Severe: If the pulp was exposed, a variety of reactions could occur, the most prevalent of which was a severe necrosis leading to development of periapical lesions. Analysis of the progressive stages of pulpal abscess and necrosis showed sprouting CGRP nerve fibers (a) at the retreating interface between abscess and vital pulp; (b) in periapical areas during onset of lesions; and (c) around chronic abscesses in granulomatous periodontal tissues. These studies are discussed in relation to various dental clinical problems such as hypersensitive teeth, episodic toothache, early onset of periapical lesions, dental anesthesia, and possible roles for sensory fibers and neuropeptides in tissue defense and healing.
Following mandibular nerve injury extensive reinnervation of sensory fibers to pulp and dentin occurs (2). A small proportion of dental nerve fibers are sympathetic as demonstrated by their cytochemistry (norepinephrine, neuropeptide Y) and by their loss following sympathetic ganglionectomy (4, 5). The sympathetic fibers primarily end on blood vessels deep in pulp. Another group of nerves which contain vasoactive intestinal peptide are more numerous than the sympathetic axons and may be parasympathetic, because they are not affected by trigeminal lesions or sympathectomy (4). The innervation of teeth is therefore complex and has diverse sensory functions, autonomic functions, and cytochemistry. During the past few years, the subgroup of dental sensory fibers that contains calcitonin gene-related peptide (CGRP), substance, P and neurokinin A has been the subject of much research (6, 7); these fibers form numerous endings in coronal pulp and dentin, along many pulpal blood vessels, and in periodontal tissue. Studies in other tissues show that CGRP and substance P are released by sensory fibers and exert profound effects that alter blood flow, inflammatory and immune responses, and connective tissue cells (8.9). Several questions can be asked about these sensory nerve fibers: What are their functions in pulpal and dentinal biology? Do they interact synergistically (or antagonistically) with nonpeptidergic sensory fibers or with autonomic fibers? What are their sensory, properties? How do they respond to injury and inflammation? What is their contribution to clinical problems such as hypersensitive dentin, toothache, and difficult anesthesia of inflamed teeth? We have recently analyzed the morphological changes of these sensory fibers to specific types of injury using immunocytochemistry for CGRP (10-13). Our previous autoradiographic studies of experimental cavities in rat molars (14) confirmed the earlier electron microscopic studies of Lilja et al. (15) demonstrating aspiration of nerve fibers into dentin under acid-etched, desiccated cavities; the effects on underlying pulp and nerve fibers were proportional to the depth of cavity and the degree of etching and air drying. From that work we devised the following injury models in rat molars: (a) Shallow cervical cavity (unetched)--This affects the sparsely innervated cervical root pulp without destroying the odontoblast layer (10). (b) Deeper cervical cavity with etching--This causes some odontoblast damage and microabscess formation followed by production of reparative dentin and healing (I 3). (c) Coronal pulp exposure leading to pulpal necrosis--Tooth reactions to pulp exposure included temporary pulp polyp formation, progressive liquefying or coagulation necrosis, and early onset of periapical lesions ( 11, 12).
The distribution of nerve fibers in tecth has been extensively documented during the past 30 yr using histological, electron microscopic, axonal transport, and immunocytochemical methods (1-3). Most of the nerve fibers pass through the roots, then make numerous branches in the coronal pulp, and terminate in the peripheral pulp or in the inner 0.1 mm of dentin. The sensory nerve fibers, which comprise the great majority of dental innervation, originate in the trigeminal ganglion and range in size from C fibers (0.1 to 1.0 um in diameter) to A-/~ (l to 5 ~m) and A-/3 fibers (6 to 10 ~m). 78
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In this article, we will review our studies concerning CGRP nerve reactions to these three types of injury. Although the CGRP (substance P, neurokinin A) nerve fibers are only one component of dental innervation, they play a principal role in neurogenic inflammation and are thus very important for pulpal and periapical pathological reactions and dental pain (16).
consent) by CGRP immunocytochemistry. The staining was most successful in teeth that were cleaved longitudinally immediately after extraction for optimal access of fixative to the pulp tissue. The immunocytochemistry procedures were similar to those used for rats (10-13), and both antirat and antihuman CGRP antibodies (Cambridge Research Biochemicals) worked well.
M A T E R I A L S AND M E T I I O D S
RESULTS
Full details of the immunocytochemical methods are given in Taylor et al. (10), Kimberly and Byers (I l), Khayat et al. (12), and Taylor and Byers (13). For all rat surgeries, the animals were deeply anesthetized with intraperitoneal Equithesin (4.25% chloral hydrate, 0.97% sodium pentobarbital) at 0.3 ml/100 g body wt. Their recovery from anesthesia was rapid and uneventful. During the postoperative period, the rats were weighed and examined daily and were found to quickly regain normal grooming, feeding, and weight gain and to exhibit an overall healthy appearance. Postinjury survival times ranged from 1 to 35 days, after which the rats were deeply anesthetized with 2.5% sodium pentobarbital (i.p.) and perfusion fixed with a mixturc of formaldehyde (4%) and picric acid (0.2%) in 0.1 M phosphate buffer. Standard immunocytochemical procedures were used as described earlier (10-13), the specificity of staining of nerve fibers was confirmed by standard controls, including omission of key reagents or preadsorption of the primary antibody with synthetic CGRP. The distribution of CGRP immunoreactive (IR) nerve fibers was analyzed in serial sections from injured and contralateral normal teeth using a Zeiss light microscope, and in some cases quantitative measurements were made. The methods for autoradiographic mapping of trigeminal dental sensory fibers (Figs. 1 and 2) are reviewed elsewhere (2). Some normal human teeth that had been extracted for orthodontic purposes were also analyzed (with the patients'
..
:~ - ~
~.=;:;:~,., ~:~" ,~.~.
FIGs 1 and 2. Autoradiographs showing the location of trigeminal sensory nerve fibers in rat molars. Most terminal branching was in the crown, with many fibers entering 0.1 mm into regular dentin (arrows), but avoiding reparative dentin (RD). Below the cervical line (dashes) very few fibers entered dentin (original magnification: Fig. 1, x87; Fig. 2, x345). Scale bars: 0.1 mm. Figure 2 is reprinted from Byers et al. (14) with permission.
Uninjured M o l a r s
The autoradiographic mapping that shows all trigeminal dental fiber types (Figs. 1 and 2) can be compared with the subgroup of CGRP-IR fibers in immunocytochemical sections of normal adult rat molars (Figs. 3 and 4). In both cases, the labeled nerve fiber bundles in the root pulp branched extensively into the coronal pulp forming the peripheral plexus of Raschkow, innervating the odontoblast layer, and entering the dentin to a distance of up to 0. I ram. There was sparse innervation of reparative dentin, and greatly reduced inncrvation in dentinal tubules of the roots, lnterradicular dentin was not innervated in normal rat molars.
Mild Injury
Four days after making shallow cavities into the cervical dentin of rat first molars, numerous CGRP-IR sprouts had formed in the underlying odontoblast layer (Figs. 5 and 6). Quantitative studies showed that the innervation from the pulp tip along the anterior mid-line pulpal surface had increased an average of more than 0.6 mm to extend into cervical root areas that normally were not innervated. Electron microscopy showed that these were new CGRP-IR nerve terminals and not preexisting nerve fibers with altered cytochemistry. The sprouting reaction had partially subsided in teeth examined 6 to 11) days postinjury and was gone by 21 days, as described by Taylor et al. (10).
FIGS 3 and 4. Immunocytochemical sections showing CGRP-IR nerve fibers branching extensively in the coronal pulp and entering into dentin for up to 0.1 mm, but avoiding reparative dentin (RD). Dashes show the cervical line (original magnification: Fig. 3, x50; Fig. 4, x130). Scale bars: 0.1 ram. Figure 4 is reprinted from Kimberly and Byers (11) with permission.
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FIGS 5 and 6. Immunocytochemical sections of a matched pair of first maxillary rat molars. In the contralateral control (Fig. 5), CGRPIR fibers were numerous near the pulp horn tip and extended along the pulp dentin border to the cervical line (dashes). The injured tooth (Fig. 6) was drilled at the cervical region 4 days earlier; numerous CGRP-IR nerve fibers have sprouted below the injury and have extended into previously sparsely innervated cervical regions along the pulp-dentin border (original magnification x80). Scale: 0.1 mm. Reprinted from Taylor et al. (10) with permission.
Microabscess Formation and Repair If the cervical cavities in rat molars were deep or were etched for 10 to 30 s with 37% phosphoric acid, microabscesses were found in the adjacent pulp 4 days later (Fig. 7) with progressive reparative dentin formation at 7 to 35 days (Figs. 8 to 10). In many cases, the injured sites were completely covered by reparative dentin by 35 days. The CGRP-IR fibers formed numerous branches in the pulp adjacent to the microabscesses. They were intermingled with the odontoblasts that were forming reparative dentin, and their sprouting subsided once the abscessed pulp had been replaced by reparative dentin, as described in Taylor and Byers (13).
Pulpal Necrosis
FIG 7. Four days after inducing the formation of an abscess (,) in cervical pulp, numerous terminal branches of CGRP-IR nerve fibers were found adjacent to the injured tissue or invading the lesion (original magnification x170). Scale: 0.1 mm
after pulpal exposure, even though much of the pulp tissue was still vital (12). In the 35-day postinjury group, when pulp tissue had been completely consumed by the advancing pulpitis, abscesses extended into the periodontal ligament and alveolar bone (Figs. 14 and 15). The CGRP-IR fibers continued to sprout around those periodontal abscesses and appeared to be actively attracted to those sites; cytochemical changes had also occurrcd by 35 days postinjury in the nearby alveolar nerves, with many more fibers staining for CGRP (ll).
Although we attempted to make pulpal exposures the same way in all cases, several reactions were found in the teeth with exposed pulp. In some, pulpal tissue was not severely damaged and had begun to form pulp polyps by 5 to 6 days postinjury (Fig. I 1); in others, a coagulation necrosis had replaced the coronal pulp by that time (Fig. 12); and in a third group liquefying necrosis was in progress (Fig. 13). Characteristic changes in the CGRP-1R nerves were found for all three reactions, all of which included sprouting of CGRP-1R fibers along the border and clustering of axons in the core of the surviving pulp. for further details see Kimberly and Byers (11) and Khayat et al. (12).
Extracted human teeth were analyzed by CGRP immunocytochemistry. In immature and mature permanent premolar or molar teeth numerous CGRP fibers were found, with many branches in the subodontoblast region, odontoblast layer, and predcntin and dentin of cervical (Fig. 16) and coronal areas.
Periapical Lesions
DISCUSSION
Quantitative measurements of periapical tissue showed that periapical lesions were beginning to develop by 5 to 8 days
The interactions between nerve fibers, pulp cells, immune cells, vasculature, and pulpal fluid are complex and dynamic.
Normal Human Teeth
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Fles 8 to 10. These teeth all received a cervical cavity that was then acid etched. An abscess (*) can be seen 7 days' postinjury (Fig. 8) with numerous CGRP-IR fibers in adjacent pulp. By 11 days postinjury (Fig. 9), large blocks of reparative dentin were forming with numerous CGRPIR fibers in the adjacent pulp. By 35 days (Fig. 10), reparative dentin had completely sealed off the injury site, and CGRP-IR fibers were less numerous and less intensely stained for CGRP-IR (original magnification x65). Scale: 0.1 mm.
FIGs 11 to 13. Three teeth showing the varied responses at 5 to 6 days after exposure of rat molar pulp, incJuding pulp polyp formation (Fig. 11), coagulation necrosis (Fig. 12), and liquefying necrosis (Fig. 13) (original magnification x80). Scale: 0.1 mm
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FIGS 14 and 15. The difference between CGGP-IR fibers in a normal rat molar periapical region (Fig. 14) and at 35 days after initiating irreversible pulpitis (Fig. 15) is shown. The abscess had extended out of the pulp into an abnormally widened periapical socket and numerous CGRP-IR sprouts were attracted toward the abscess (,). In normal tissue only a few nerves stained for CGRP-IR are seen, compared with the numerous CGRP-IR axons near abscessed ligament (original magnification x45). Scale: 0.1 mm. Reprinted from Kimberly and Byers (11 ) with permission.
NERVES SENSORY (non-PeDtide) (peptides)
SYMPATHETIC & ParasymD
>ULP CELLS ..Odontoblasts 9Fibroblast s Immune Cells
FLUID DYNAMICS PulDal Oentinal Vascular
"Three Worlds* M.C.Escher (1955)
F~G 16. Human permanent left maxillary first premolar with numerous CGRP-IR nerve fibers in the subodontoblast region, in the odontoblast layer (=), and in dentin (D) (original magnification x190). Scale: 0.1 mm.
FIG 17. M. C. Escher's image "Three Worlds" (1955) has been used here to illustrate the point that various dental nerve fibers, pulp cells, and factors affecting fluid dynamics in teeth interact in numerous complex ways that can activate pain sensations (represented by the fish). It is a challenge for dental research to define those interactions and to devise optimal preventive measures and pain management techniques. Repnnted with permission from M. C. Escher Heirs, 9 1988 M. C. Escher Heirs/Cordon Art-Baarn, Holland.
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Clearly, some combinations of those interactions cause pain and others do not. Although there is still much to learn about neural and cellular interactions in pulp patholosis, several general statements can be made based on clinical observations (I 7), on numerous experiments by Br~instr6m (18) and others, and on our series of immunocytochemical experiments in rats.
Hypersensitive Dentin and Toothache Many experiments (e.g. 18-20) have suggested that fluid movement in dentinal tubules that are open to the oral environment can cause dental hypersensitivity by stimulating nerve fibers along the pulp-dentin border. Our recent experiments and those by Plackova (21) suggest that sprouting of dental nerve fibers may also have occurred; if so, then the hydrodynamic stimuli to exposed dentin would activate many more nerve fibers than normal. The neural morphological changes were dynamic, with rapid increases in terminal branching and reversion to normal over a period of days. Such temporary fluctuations in neural density may affect the duration of tooth hypersensitivity, as can progressive occlusion of the exposed dentinal tubules (22). Similarly, episodic toothache may include neural fluctuations, with cycles of increased nerve fibers and peptide cytochemical alteration followed by decreases, perhaps a~ociated with cycles of abscess expansion and pulpal healing. Recent immunocytochemical studies of human teeth suggest that there may be sprouting of dental nerves and altered neuropeptides associated with painful caries (23), but more immunocytochemical work needs to be done concerning histopathological correlations with clinical pain.
Dental Anesthesia A regional nerve block frequently fails to affect an inflamed tooth even though adjacent healthy teeth are well anesthetized (24). Our observation that neuropeptides such as CGRP were elevated in trunk axons of trigeminal nerves that innervated inflamed tissue (11~ suggests that those fibers may have an altered capacity for anesthesia due to cytochemical changes extending throughout the affected nerve fibers. That suggestion is supported by recent studies showing rapid alterations in neuropeptide mRNAs in dorsal root ganglia and spinal cord neurons within hours of inducing inflammation (25). Since immunocytochemistry shows relatively large chemical changes for trigeminal axons, one could certainly postulate subtle changes in membrane molecular structure that would affect anesthesia sensitivity; but further work is needed to determine whether such changes occur.
Periapical Lesions Our studies ofperiapical lesions found significant widening of the socket and sprouting of CGRP-IR periapical fibers when vital pulp was still present (12). This suggested that neuropeptides and sensory nerve fiber branching may have contributed to lesion development. Other etiologic factors such as diffusion of bacterial products from the pulpal abscesses may also have contributed. Involvement of a neurogenie mechanism in the onset of periapical lesions is sup-
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ported by evidence for stimulation of blood flow, inflammatory reactions, immune cell activity, and other cellular functions by neuropeptides (8, 9, 26). Much still remains to be determined concerning the role of neuropeptides and neural reactions in tissue defense and healing. Although the inflammatory actions of some sensory nerve fibers are well recognized (26), we should perhaps consider those nerve fibers to be activated following injury primarily for facilitation of inflammatory reactions and tissue healing, with sensory activity as a secondary function. Recent studies of other dental injury models such as orthodontic displacement in rats (27) give further evidence of injuryinduced changes in neural structure, suggesting that such changes may be common to most (or all) dental injuries. The evidence for CGRP innervation of human teeth (Fig. 16) suggests that information gained from animal experiments will have general relevance for human dental biology and pathology. Evidence for interactions between peptidergic sensory fibers and sympathetic fibers in pathologic reactions comes from studies of experimental arthritis (25, 26) or of injured optic tissues (28) in which neural sprouting or neuropeptide changes were associated with both sensory and sympathetic nerve reactions. A full understanding of neural reactions to dental injury and inflammation should therefore include studies of sympathetic, parasympathetic, and nonpeptidergic sensory fibers as well as of the peptidergic sensory fibers and their interactions with pulp (Fig. 17). Finally, evidence for complex interactions between macrophages, leukocytes, and neuropeptides (9) includes stimulation of nerve growth factor production (29), suggesting that nerve growth factor may be a critical factor in controlling the nerve sprouting reactions to dental injury and inflammation (30). This research was supported by NIH Grant OE 05159 to Dr. Byers end by NIH Grant 2-S07-RR05346 (BSRG) and the Graduate Endodontics Fund, University of Washington. Drs. C. L. Kimbedy and P. E. Taylor received salary support from the U.S. Navy. Dr. B. G. Khayat received partial salary support from NIH Grant AG 00122. We thank Dr. G. Harrington, Dr. R. Oswald, and Mr. P. E. Redd for helpful discussions. We also thank Kelly B MecJfl for exten,wve expert technical assistance, Diane Ryba for photographic work, and Nancy Sutton for secretarial work. Our experiments conformed to guidelines of the Human Subjects and Animal Care Committees of the University of Washington. Dr. Byers is a research professor. Departments of Anesthesiology, Endodont~s, and Biologm_,alStructure, University of Washington, Seattle, WA. Drs. Khayat, Kimberly, and Taylor are affiliated with the Department of Endodontics, University of Washington. Address requests for reprints to Dr. Byers, Department of Anesthesiolngy, RN-10, University of Washington, Seattle, WA 98195.
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7. Silverman JD, Kruger L. Calcitonin-gene-retated-peptide immunoreact}ve innervation of the rat head with empelasis on specialized sensory structure. J Comp Neurol 1989;280:303-30. 8. Brain SD, Williams TJ, Tippins JR, Morris HR, Maclntyre I. Calcitonin gene-ralated peptide is a potent vasodilator. Nature 1985;313:54-6. 9. Payan DG, McGillis JP, Renold FK, Mitsuhashi M, Goet.zl EJ. The role of brain peptides in neuroimmunomodulation. Ann NY Acad Sci 1987;496:18291. 10. 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. 11. Kimberly CL, Byers MR. Inflammation of rat molar pulp and penodontium causes increased calcitonin gene related pepede and axonal sprouting. Anat Rec 1988;222:289-300. 12. Khayat BG, Byers MR, Taylor PE, Mecifi KB, Kimberly CL Responses of nerve fibers to pulpal inflammation and ponapical lesions in rat molars demonstrated by calcitonin gene-related peptide immunocytochemistry. J Endodon 1988;14:577-87. 13. Taylor PE, Byers MR. Response of CGRP-immunoreactive nerve fibers to limited pulpitis and healing in rat molars. Arch Oral Biol (in press). 14. Byers MR, Narhi MVO, Mecifi KB. Acute and chronic reactions of dental sensory nerve fibers to cavities and desiccation in rat molars. Anat Rec 1988;221:872-83. 15. IJIja J, Nordenvall KJ, Brannstrom M. Dentin sensitivity, odontoblasts and nerves under desiccated or infected experimental cavities. Swed Dent J 1982;6:93-103. 16. Olgart LM. The role of local factors in dentin and pulp intradental pain mechanisms. J Dent Res 1985;64(spe~al issue):572-8 17. Trowt)ridge HO. Intradental sensory units: physK)k~jlcal and clinKT,al aspects. J Endodon 1985;11:489-98. 18. Brannstr0m M. Dentin and pulp in restorative dentistry. London: Wolf Medical Publications, 1981.
Joumal of Endodontics 19. Karlsson UL, Penney DA. Natural desensitization of exposed tooth roots in dogs J Dent Res 1975:54:982-6. 20. Hirvonen TJ, Narhi MVO, Hakumaki MDK. The excital~lity of dog ;~JIp nerves in relation to the condition of dentin surface. J Endodon 1984;10: 294-8. 21. Plackova A. Pathologic changes in the innervation of the dental pulp during carious process. J Dent Res 1966;45:62-5. 22. Pashley D. Dentin-predentin complex and its permeability: physiologic overview. J Dent Res 1985;64(speoal issue):613-20. 23. Yamaura Y. Immunohistochemc, al investigations on the nerve fibers of the dental pulp after the cavity preparation. Jpo J Conserv Dent 1987;30:82438. 24. Brown RD. The failure of IocaJ anesthesia in acute inflammation. Br Dent J 1981;151:47-51. 25. ladarola MJ, Draisoi G. Elevation of spinal cord dynorp~in mRNA compared to dorsal root ganglio~ beptide mRNAs during peripheral inflammation. In: Bessen JM. Guilbaud G, eds. The arthritic rat as a model of clinkcal pare? New York: Elsevier, 1988;173-83. 26. Levine JD, Coderre T J, Basbaum AI. The peripheral nervous system and the inflammatory process. Pain Res Clin Managem 1988:3:33-43. 27. Kvinnsland I, Kvinnsland S. Changes in CGRP-immunoreact/ve nerve fibers during experimental tooth movement in rats. Eur J O ~ (in press). 28. Tererlghi G, Zhen(j SQ, Unger WG, Polak J. Morphological changes of sensory CGRP-immunoreactive and sympathetr nerves in peripheral tissues following chronic denervation. Histochemistry 1986;86:89-95. 29. IJndhotm D, Heumann R, Meyer M, Thoenen H. Intedeukin-1 regulates synthesis of nerve growth factor in non-neuronal cells of rat sciatic nerve. Nature 1987;330:658-9. 30. Byers MR, Bothwell MA, Mecifi KB, Schatteman GC. InteractK)ns of nerves and pulp cells in normal and injured teeth analysed by LM and EM immunocytochemistry for NGF-receptor. Neurosci Abstracts 1988;14:1169.
