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An autoradiographic demonstration of trigeminal nerve terminations in the rat tooth
SCIENTIFIC ARTICLES
An autoradiographic
demonstration
trigeminal nerve terminations
of
in t h e r a t t o o t h
Richard A. Menke, DDS, MS; Franklin S. Weine, DDS, MSD: Philip S. Ullrm~, PhD; a n d M a r s h a l l H. S m u l s o n , DDS, Chicaqo
By u s i n g a n e u r o a n a t o m i c m e t h o d , triqemin~l n e r v e t e r m i n a tions w e r e s h o w n in the m i d d l e dentin, i n n e r dentin, predentin, a n d s u b o d o n t o b l a s t i c zone, but not in the outer dentin, odontoblastic layer, or pulp h o r n of rat molars. A p i c a l n e r v e t e r m i n a t i o n s in rat incisors w e r e f o u n d in the predentin, s u b o d o n t o b l a s t i c zone, a n d pulp, but not ir~ the h y p o c a l c i fled e n a m e l , dentin, or odontoblastic l a y e r s .
The problem of dentinal sengitivity has defied solution for many years, principally because investigators have used different experimental techniques. Physiologic experiments l"e denied the existence of functional, sensory dentinal innervation because specific stimuli could not diffuse to, or raise, the sensory threshold of dentinal nerves.
Nerves have been identified in the predentin and, on occasion, even in mature dentin by using the light microscope. T M By using the electron microscope, nerve terminations were found attached to the odontoblastic process in predentin and dentin. 11-~7 These terminations contained neuro-
128
tubules, microvesieles, and mitochondria; they also were noticed in the formation of dentin. TM The questions raised from these different experiments are: What is the origin of dentinal nerves seen by histologists? Where do they terminate? Are they functional trigeminal afferent terminations? Neuroanatomists ta25 demonstrated that if a radioactive amino acid were injected into the cell body of a neuron, the cell body would synthesize a protein from the amino acid and transport it by the fast-axon transport system to the neuronal terminations. By using autoradiographic grain counts, the location of labeled amino acids would show the termination of these functioning neurons, if investigators used short postinjection survival times. This neuroanatomic method was used to answer the questions raised by other dental investigators. Methods and Materials The tracer used was tritiated L4-5 leucine (20/xCi//.d-concentration). After anesthetizing six 100-day-old female Sprague-Dawley rats intraperitoneally with Nembutal, the right trigeminal ganglion was surgically exposed. By using a syringe,* 2g,l of 3H leucine was injected 1 mm caudally to the right lateral protuberance of the mandibular branch at a
rate of 0.1/zl/min. Animals were killed from one to five hours after injection. The experimental right ganglion and right side of the mandible and the control left ganglion and left side of the mandible were removed and fixed for 72 hours in 10% Formalin. Each side of the mandible contained three molars and an apical developing incisor. The mandibles were decalcified in formic acid and sodium citrate. All specimens were washed, dehydrated, embedded in paraffin, sectioned at 7/zm, and mounted on slides. The slides then were deparaffinized and immersed in an emulsion; placed in light, tight boxes; and stored at 0 C. After optimal exposure, all slides were placed in developert at 20 C for three minutes, washed in acetic acid, and placed in fixativet at 20 C for three minutes. The ganglia were stained with cresyl violet, and the mandibles were stained with Harris's hematoxylin and eosin. Autoradiographic mean silver grain counts were used to compare the right experimental side with the left control side. To find mean silver grain counts, an eyepiece grid with a reticular pattern formed by 100 square-micron squares was u s e d under an oil emersion objective. Five slides were selected from the mandibular right side of each animal and five
JOURNAL OF ENDODONTICS [ VOL 3. NO 4. APRIl, 1977
from the mandibular left side. There were five sections per slide, and the middle section on each slide was used for counting. In each of the three molars, three counts were made per section by randomly counting two adjacent squares in the outer dentin, middle dentin, inner dentin, predentin, odontoblastic layer, subodontoblastic zone, and pulp. In the apical developing incisor, three counts were made per section by randomly counting two adjacent squares in the hypocalcified enamel, dentin, predentin, odontoblastic layer, subodontoblastic zone, and pulp. Thus, 15 counts were made for each layer of each tooth of each animal, both right and left, and mean silver grain counts were determined. The observer did not know which animal or which side he was counting. Counts were made for 30-minute intervals with at least a four-hour rest period between counting sessions to avoid eyestrain. Student's t tests were used to evaluate statistically if mean silver grain counts on the experimental side were from the same or from different populations as mean silver grain counts on the background of the control side. Results
All trigeminal right mandibular cell bodies showed incorporation of radioactive leucine and synthesis of protein in the cell bodies (Fig 1,2). These radioactive proteins were transported to their afferent trigeminal nerve terminations in the teeth of the right side of the mandible only. Figures 3A and 4A show the right molar and incisor containing silver grains. The left ganglion (Fig 2B), the molars (Fig 3B), and the incisors ( F i g 4 B) contained silver grains only in the background. Graphs were made of each specific layer studied. Student's t values were plotted on the ordinate, and postinjec-
Fig 1--Lateral protuberance of typical injected trigeminal ganglion. Radioactive leucine appears as black silver grains in somata. Most cell bodies are completely filled with isotope (cresyl violet, •
Fig 2,4--Higher magnification of mandibular cell bodies of experimental right trigeminal ganglion. ,4rrow shows cell body heavily labeled with radioactive leucine (cresyl violet, x405). 2B--Mandibular cell bodies of control left trigeminal ganglion from same animal as experimental right trigeminal ganglion in Figure 2,4. Arrow shows typical histologic pattern of somata. Notice only background radiation (cresyl violet, •
tion survival times on the abscissa; t values greater than 2.76 were statistically significant when P--0.01. In comparing the mean number of silver grains over areas with no tissue on the experimental side to the mean number of silver grains over similar areas on the control, t values are plotted on G r a p h 1 as "slide background." There were no significant t values. These counts served as controls for the counting and autoradiographic procedures. Incisor enamel ( G r a p h 2) also served as a control. The third molars of animals killed two and three hours after the injection were not used because of errors in sectioning. In the molars, the areas showing no nerve terminations, or where no significant t values were calculated, were in the outer dentin ( G r a p h 3), the odontoblastic layer ( G r a p h 4), and the pulp (Graph 5). The areas showing nerve termina-
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Fig 4A--Microscopic appearance o] apical portion o/mandibular incisor oJ animal killed/our hours after injection. Notice evidence of radioactive leucine (silver grains) in predentinal nerve terminations at pulpodentinal membrane, which are transported there by Jast-axon transport system (H&E, x 768). 4 B - Incisor [rom control side o] same animal as incisor in Figure 4A. Notice only background silver grains (H&E, x768). E, hypocalci]ied enamel; D, dentin; P, predentin; O, odontoblastic layer; S, subodontoblastic zone.
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Fig 3A---Pulp horn o] second molar o/animal killed/our hours alter injection. Notice evidence of radioactive leucine (silver grains) [rom trigeminal cell bodies in nerve terminations o[ dentin and predentin (H&E, x 768). 3B--Second molar ]rom control side o] same animal as molar in Figure 3A. Only background silver grains are present (H&E, x768). M, middle dentin; I, inner dentin; P, predentin; O, odontoblastic layer. 130
tions in the molars were determined. The junction of the inner-third dentin and middle-third dentin (termed middle dentin, G r a p h 6) showed significant t values for the first molar at two and three hours, all molars at four hours, and the second and third molars at five hours. The inner dentin had significant t values in all molars in the animals killed four and five hours after the injection ( G r a p h 7). In the predentin, significant t values were determined for the first molar at four hours and the second molar at five hours ( G r a p h 8). Statistically notable t values were found in the subodontoblastic zone ( G r a p h 9) of the second and third molars in animals killed four and five hours after the injection. In the incisor, there were no trigeminal nerve terminations found in
the enamel ( G r a p h 2), the inner dentin ( G r a p h 7), or the odontoblastic layer ( G r a p h 4). In the predentin of the incisor at the pulpodentinal membrane, significant t values were determined at two, four, and five hours ( G r a p h 8). Statistically significant t values were found in the subodontoblastic zone of an incisor at two hours ( G r a p h 9). The pulp showed significant t values in incisors at two, four, and five hours ( G r a p h 5). The data are summarized in the Table, which lists the layers studied. "Plus" signs indicate that on the injected side, the layer had a statistically higher silver grain count than the same area on the control side. These areas presumably contain nerve terminations of the peripheral processes of cell bodies located in the trigeminal ganglion.
JOURNAL OF ENDODONTICS I VOL 3, NO 4, APRIL 1977
Slide Background 3.00 P=.OI
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Graph I - - B a c k g r o u n d of slide served as control for counting and autoradiographic procedures. Notice that there are no significant t values above 2.76.
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Graph 3 - - O u t e r dentin did not show any significant t values when experimental side was compared with control side.
Cariness and Barkley 26 found no significant variation for silver grain counts with tritium as a label if the sections were of the same thickness and mounted on separate slides. Sawicki, Blaton, and Rowifiski2~ found that background counts should be calculated from tissue similar to experimental tissue. To evaluate two means statistically, t values were used. W h e n Pffi0.01, we can state with almost absolute ( 9 9 % ) confidence that the two means are from different populations. Therefore, t values greater than 2.76 show statistically significant increased silver grains in the experimental right layers of teeth and, hence, the presence of transported 3H leucine from the trigeminal ganglia to the peripheral nerve terminations. The presence of dentinal nerve terminations has been denied by some researchers z-5 because they were unable to excite any dentinal nerves with chemical stimuli. This discrepancy may have stemmed from the fact that the solution they used did not diffuse through the dentinal tubules to excite afferent nerves in the inner and predentinal layers. Thus, their techniques do not warrant statements about terminations. Some investigators studied nerve fibers with the light microscope but could not interpret exactly where the fibers terminatedF -lo Others used the electron microscope and described nerve terminations in the dentin that had cytologic characteristics identical to nerve terminations described in other locations. 1z-z8 The current work is consistent with the electron microscopic studies on nerve terminations. Only the nerve terminations were studied by using an accepted neuroanatomic method for locating such terminations.19-25 The rat incisor is a continually erupting tooth that shows continuously developing enamel and dentin at its 131
JOURNAL OF ENDODONTICS I VOt 3, NO 4, A n ' ~
apical inferior border3 8 Trigeminal nerve terminations were found in the predentin that would have become the middle dentin if the tooth had matured. Nerve terminations also were found in the subodontoblastic zone and the pulp of this immature tooth. The rat molar has been used by many investigators because of its similarity to the human molar. 28 In this study, no trigeminal nerve terminations were found in the pulp inferior to the subodontoblastic zone of the pulp horn. Th e subodontoblastic zone did contain trigeminal nerve endings; this is consistent with other studies. 15,18 The odontoblastic layer did not contain any trigeminal nerve terminations. Investigators who used the electron microscope have shown nerve axons to pass between odontoblasts, but were unable to show a direct contact between nerve fibers and odontoblasts. T M In addition, these experimenters found an attachment between nerve fibers and the odontoblastie process in the predentin and inner dentin but not in the outer dentin. In this study, trigeminal nerve terminations were noticed in these same areas. These terminations are functional nerve endings receiving a precursory protein from their cell body. This study raises several questions: Are the structures ascending in the layers one nerve with branches or terminations to each layer, or are they each a separate nerve to each layer? Are the subodontoblastic zone nerve terminations from myelinated nerve fibers and the dentinal nerves from unmyelinated nerves? Do the nerves attached to the odontoblastic process in dentin respond only to stimuli that affect the osmotic pressure, flow of fluid, a n d / o r membrane of the odontoblastic process? Do the nerve terminations in the subodontoblastic zone respond only to stimuli that change the intrapulpal pressure? Are there any efferent sympathetic nerve terminations in the dentin? 132
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Graph 6--1unction o f middle and inner dentin was termed middle dentin. Significant t values were noticed in /irst molar at two and three hours, all molars at [our hours, and second and third molars at five hours.
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Summary
Reterences
A neuroanatomic method using the fast-axon transport of a radioactive isotope showed the termination of the trigeminal nerve in rat molars and incisors. Autoradiographic mean silver grain counts of experimental and control teeth were compared using Student's t tests. Statistically significant t values ( P = 0 . 0 1 ) showed that trigeminal nerve terminations were found in the middle dentin, inner dentin, predentin, and subodontoblastic zone, but not in the outer dentin, odontoblastic layer, or pulp of the rat molars. In the apical rat incisor, nerve terminations were found at the pulpodentinal membrane, subodontoblastic zone, and pulp, but not in the enamel, dentin, or odontoblastic layers.
1. Anderson, D.J.; Curwen, M.P.; and Howard, L.V. The sensitivity of human dentin. J Dent Res 37:669 Aug 1958. 2. Anderson, D.J., and Naylor, M.N. Chemical excitants of pain in human dentine and dental pulp. Arch Oral Biol 7:413 May-June 1962. 3. Anderson, D.J., and Matthews, B. An investigation into the reputed desensitizing effect of applying silver nitrate and strontium chloride to human dentine. Arch Oral Biol 11:1129 Nov 1966. 4. Dellow, P.G., and Roberts, M.L. Bradykinin application to dentine; a study of a sensory receptor mechanism. Aust Dent J 11:384 Dec 1966. 5. Brannstr6m, M., and Astr6m, A. A study on the mechanism of pain elicited from the dentin. J Dent Res 43:619 July-Aug 1964. 6. Van Hassel, H.J., and Ervin, M. Correlation of sensory response with intrapulpal temperature and pressure. J Dent Res 53:144 Feb 1973. 7. Held, A.J., and Baud, C.A. The innervation of the dental organ. Oral Surg 8:1262 Dec 1955. 8. Hattyasy, D. Continuous regeneration of the dentinal nerve-endings. Nature 189:72 Jan 7, 1961. 9. L6rfinth, C., and Csfinyi, K. Innervation of teeth. Acta Morph Acad Sci Hung 15:391, 1967. 10. Fearnhead, R.W. The histological demonstration of nerve fibres in human dentine. In Anderson, D.J., ed. Sensory mechanisms in dentine. New York, MacMillan Co., 1963, p 15. 11. Arwill, T. The ultrastructure of the pulpo-dentinal border zone. In Symons, N.B.B., ed. Dentine and pulp: their structure and reactions. London, Churchill Livingstone, 1968, p 147. 12. Frank, R.M. Etude au microscope electronique de l'odontoblaste et du canalicule dentinaire humain. Arch Oral Biol 11:179 Feb 1966. 13. Frank, R.M. Attachment sites between the odontoblast process and the intradentinal nerve fibre. Arch Oral Biol 13:833 July 1968. 14. Frank, R.M. Ultrastructural relationship between the odontoblast, its process and the nerve fibre. In Symons, N.B:B., ed. Dentine and pulp: their
*Hamilton Industries, Two Rivers, Wis. "~Eastman Kodak Co., Rochester, NY. This paper was presented at the annual meeting of the American Association of Endodontists on April 14, 1974, in San Diego, Calif; its authors were the recipients of the Endodontic Research Award. This article is derived from a thesis submitted by Dr. Menke to the faculty of the graduate school of Loyola University of Chicago in partial fulfillment of the requirements for the master of science degree. Dr. Menke is in private practice limited to endodontics in Worthington, Ohio. Dr. Weine is professor and director, postgraduate endodontics, Loyola University of Chicago, Maywood, IlL Dr. Ulinski is assistant professor, department of anatomy, University of Chicago. Dr. Smulson is professor and chairman, department of endodontics, Loyola University of Chicago. Requests for reprints should be directed to Dr. F. Weine, School of Dentistry, Loyola University of Chicago, 2160 S First Ave, Maywood, I11 60153.
134
structure and reactions. London, Churchill Livingstone, 1968, p 115. 15. Frank, R.M.; Sauvage, C.; and Frank, P. Morphological basis of dental sensitivity. Int Dent J 22:1 March 1972. 16. Johansen, E. Ultrastructure of dentine. In Miles, A.E.W., ed. Structural and chemical organization of teeth. New York, Academic Press Inc., 1967, vol 11, p 35. 17. Roane, J.B.; Foreman, D.W.; Melfi, R.C.; and Marshall, F.J. An ultrastructural study of dentinal innervation in the adult human tooth. Oral Surg 35:94 Jan 1973. 18. Corpron, R.E., and Avery, J.K. The ultrastructure of intradental nerves in developing mouse molars. Anat Rec 175:585 March 1973. 19. Droz, B., and Leblond, C.P. Axonal migration of proteins in the central nervous system and peripheral nerves as shown by radioautography. J Comp Neurol 121:325 Dec 1963. 20. Lasek, R. Protein transport in neurons. Int Rev Neurol 13:289, 1970. 21. Lasek, R. Axoplasmic transport in cat dorsal root ganglion cells: as studied with [3-H]-L-leucine. Brain Res 7:360 March 1968. 22. Sj6strand, J. Rapid axoplasmic transport of labelled proteins in the vagus and hypoglossal nerves of the rabbit. Exp Brain Res 8:105, 1969. 23. Schonback, J., and Cu6nod, M. Axoplasmic migration of protein. A light microscopic autoradiographic study in the avian retino-tectal pathway. Exp Brain Res 12:275, 1971. 24. Lasek, R.; Joseph, B.S.; and Whitlock, D.G. Evaluation of a radioautographic neuroanatomic tracing method. Brain Res 8:319 May 1968. 25. Cowan, W.M.; Gottlieb, D.I.; Hendrickson, A.E.; Price, J.L.; and Woolsey, T.A. The autoradiographic demonstration of axonal connections in the central nervous system. Brain Res 37:21 Feb 11, 1972. 26. Cariness, V.S., and Barkley, D.S. Section thickness and grain count variation in autoradiographic tritium. Stain Technol 46:131 May 1971. 27. Sawicki, W.; Blaton, O.; and Rowifiski, J. Correction of autoradiographic grain count in respect to precisely calculated background. Histochemie 26:67, 1971. 28. Schour, I., and Massler, M. The teeth. In Farris, E.J., ed. The rat in laboratory investigation. New York, Hafner Publishing Co., 1962, p 104.
An autoradiographic
demonstration
trigeminal nerve terminations
of
in t h e r a t t o o t h
Richard A. Menke, DDS, MS; Franklin S. Weine, DDS, MSD: Philip S. Ullrm~, PhD; a n d M a r s h a l l H. S m u l s o n , DDS, Chicaqo
By u s i n g a n e u r o a n a t o m i c m e t h o d , triqemin~l n e r v e t e r m i n a tions w e r e s h o w n in the m i d d l e dentin, i n n e r dentin, predentin, a n d s u b o d o n t o b l a s t i c zone, but not in the outer dentin, odontoblastic layer, or pulp h o r n of rat molars. A p i c a l n e r v e t e r m i n a t i o n s in rat incisors w e r e f o u n d in the predentin, s u b o d o n t o b l a s t i c zone, a n d pulp, but not ir~ the h y p o c a l c i fled e n a m e l , dentin, or odontoblastic l a y e r s .
The problem of dentinal sengitivity has defied solution for many years, principally because investigators have used different experimental techniques. Physiologic experiments l"e denied the existence of functional, sensory dentinal innervation because specific stimuli could not diffuse to, or raise, the sensory threshold of dentinal nerves.
Nerves have been identified in the predentin and, on occasion, even in mature dentin by using the light microscope. T M By using the electron microscope, nerve terminations were found attached to the odontoblastic process in predentin and dentin. 11-~7 These terminations contained neuro-
128
tubules, microvesieles, and mitochondria; they also were noticed in the formation of dentin. TM The questions raised from these different experiments are: What is the origin of dentinal nerves seen by histologists? Where do they terminate? Are they functional trigeminal afferent terminations? Neuroanatomists ta25 demonstrated that if a radioactive amino acid were injected into the cell body of a neuron, the cell body would synthesize a protein from the amino acid and transport it by the fast-axon transport system to the neuronal terminations. By using autoradiographic grain counts, the location of labeled amino acids would show the termination of these functioning neurons, if investigators used short postinjection survival times. This neuroanatomic method was used to answer the questions raised by other dental investigators. Methods and Materials The tracer used was tritiated L4-5 leucine (20/xCi//.d-concentration). After anesthetizing six 100-day-old female Sprague-Dawley rats intraperitoneally with Nembutal, the right trigeminal ganglion was surgically exposed. By using a syringe,* 2g,l of 3H leucine was injected 1 mm caudally to the right lateral protuberance of the mandibular branch at a
rate of 0.1/zl/min. Animals were killed from one to five hours after injection. The experimental right ganglion and right side of the mandible and the control left ganglion and left side of the mandible were removed and fixed for 72 hours in 10% Formalin. Each side of the mandible contained three molars and an apical developing incisor. The mandibles were decalcified in formic acid and sodium citrate. All specimens were washed, dehydrated, embedded in paraffin, sectioned at 7/zm, and mounted on slides. The slides then were deparaffinized and immersed in an emulsion; placed in light, tight boxes; and stored at 0 C. After optimal exposure, all slides were placed in developert at 20 C for three minutes, washed in acetic acid, and placed in fixativet at 20 C for three minutes. The ganglia were stained with cresyl violet, and the mandibles were stained with Harris's hematoxylin and eosin. Autoradiographic mean silver grain counts were used to compare the right experimental side with the left control side. To find mean silver grain counts, an eyepiece grid with a reticular pattern formed by 100 square-micron squares was u s e d under an oil emersion objective. Five slides were selected from the mandibular right side of each animal and five
JOURNAL OF ENDODONTICS [ VOL 3. NO 4. APRIl, 1977
from the mandibular left side. There were five sections per slide, and the middle section on each slide was used for counting. In each of the three molars, three counts were made per section by randomly counting two adjacent squares in the outer dentin, middle dentin, inner dentin, predentin, odontoblastic layer, subodontoblastic zone, and pulp. In the apical developing incisor, three counts were made per section by randomly counting two adjacent squares in the hypocalcified enamel, dentin, predentin, odontoblastic layer, subodontoblastic zone, and pulp. Thus, 15 counts were made for each layer of each tooth of each animal, both right and left, and mean silver grain counts were determined. The observer did not know which animal or which side he was counting. Counts were made for 30-minute intervals with at least a four-hour rest period between counting sessions to avoid eyestrain. Student's t tests were used to evaluate statistically if mean silver grain counts on the experimental side were from the same or from different populations as mean silver grain counts on the background of the control side. Results
All trigeminal right mandibular cell bodies showed incorporation of radioactive leucine and synthesis of protein in the cell bodies (Fig 1,2). These radioactive proteins were transported to their afferent trigeminal nerve terminations in the teeth of the right side of the mandible only. Figures 3A and 4A show the right molar and incisor containing silver grains. The left ganglion (Fig 2B), the molars (Fig 3B), and the incisors ( F i g 4 B) contained silver grains only in the background. Graphs were made of each specific layer studied. Student's t values were plotted on the ordinate, and postinjec-
Fig 1--Lateral protuberance of typical injected trigeminal ganglion. Radioactive leucine appears as black silver grains in somata. Most cell bodies are completely filled with isotope (cresyl violet, •
Fig 2,4--Higher magnification of mandibular cell bodies of experimental right trigeminal ganglion. ,4rrow shows cell body heavily labeled with radioactive leucine (cresyl violet, x405). 2B--Mandibular cell bodies of control left trigeminal ganglion from same animal as experimental right trigeminal ganglion in Figure 2,4. Arrow shows typical histologic pattern of somata. Notice only background radiation (cresyl violet, •
tion survival times on the abscissa; t values greater than 2.76 were statistically significant when P--0.01. In comparing the mean number of silver grains over areas with no tissue on the experimental side to the mean number of silver grains over similar areas on the control, t values are plotted on G r a p h 1 as "slide background." There were no significant t values. These counts served as controls for the counting and autoradiographic procedures. Incisor enamel ( G r a p h 2) also served as a control. The third molars of animals killed two and three hours after the injection were not used because of errors in sectioning. In the molars, the areas showing no nerve terminations, or where no significant t values were calculated, were in the outer dentin ( G r a p h 3), the odontoblastic layer ( G r a p h 4), and the pulp (Graph 5). The areas showing nerve termina-
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'
9
Fig 4A--Microscopic appearance o] apical portion o/mandibular incisor oJ animal killed/our hours after injection. Notice evidence of radioactive leucine (silver grains) in predentinal nerve terminations at pulpodentinal membrane, which are transported there by Jast-axon transport system (H&E, x 768). 4 B - Incisor [rom control side o] same animal as incisor in Figure 4A. Notice only background silver grains (H&E, x768). E, hypocalci]ied enamel; D, dentin; P, predentin; O, odontoblastic layer; S, subodontoblastic zone.
t. 9
9
It
Fig 3A---Pulp horn o] second molar o/animal killed/our hours alter injection. Notice evidence of radioactive leucine (silver grains) [rom trigeminal cell bodies in nerve terminations o[ dentin and predentin (H&E, x 768). 3B--Second molar ]rom control side o] same animal as molar in Figure 3A. Only background silver grains are present (H&E, x768). M, middle dentin; I, inner dentin; P, predentin; O, odontoblastic layer. 130
tions in the molars were determined. The junction of the inner-third dentin and middle-third dentin (termed middle dentin, G r a p h 6) showed significant t values for the first molar at two and three hours, all molars at four hours, and the second and third molars at five hours. The inner dentin had significant t values in all molars in the animals killed four and five hours after the injection ( G r a p h 7). In the predentin, significant t values were determined for the first molar at four hours and the second molar at five hours ( G r a p h 8). Statistically notable t values were found in the subodontoblastic zone ( G r a p h 9) of the second and third molars in animals killed four and five hours after the injection. In the incisor, there were no trigeminal nerve terminations found in
the enamel ( G r a p h 2), the inner dentin ( G r a p h 7), or the odontoblastic layer ( G r a p h 4). In the predentin of the incisor at the pulpodentinal membrane, significant t values were determined at two, four, and five hours ( G r a p h 8). Statistically significant t values were found in the subodontoblastic zone of an incisor at two hours ( G r a p h 9). The pulp showed significant t values in incisors at two, four, and five hours ( G r a p h 5). The data are summarized in the Table, which lists the layers studied. "Plus" signs indicate that on the injected side, the layer had a statistically higher silver grain count than the same area on the control side. These areas presumably contain nerve terminations of the peripheral processes of cell bodies located in the trigeminal ganglion.
JOURNAL OF ENDODONTICS I VOL 3, NO 4, APRIL 1977
Slide Background 3.00 P=.OI
2.76
Graph I - - B a c k g r o u n d of slide served as control for counting and autoradiographic procedures. Notice that there are no significant t values above 2.76.
2.00
1.00
Discussion
@
).OC 2
4
3
5
Hours post injection
Incisor enamel 3.00 P=.01
2.76 2.00
(n
Iraph 2 - - E n a m e l o / i n kor served as control ~r technique and towed no significant t dues.
-~ -~ > ~"
1.00 o
@ 0.00 2
o
3
4
Hours post i n j e c t i o n
Outer dentin 3.00
o
I 't m o l a r
a
2.d m o l a r
A
3 rd m o l a r
.P=.01
2.76 2.00
o rl
1.00 13 A
0.00
I
rt
@ A
o
O @
O
2
3
0
A
D
4
Hours post injection
5
Graph 3 - - O u t e r dentin did not show any significant t values when experimental side was compared with control side.
Cariness and Barkley 26 found no significant variation for silver grain counts with tritium as a label if the sections were of the same thickness and mounted on separate slides. Sawicki, Blaton, and Rowifiski2~ found that background counts should be calculated from tissue similar to experimental tissue. To evaluate two means statistically, t values were used. W h e n Pffi0.01, we can state with almost absolute ( 9 9 % ) confidence that the two means are from different populations. Therefore, t values greater than 2.76 show statistically significant increased silver grains in the experimental right layers of teeth and, hence, the presence of transported 3H leucine from the trigeminal ganglia to the peripheral nerve terminations. The presence of dentinal nerve terminations has been denied by some researchers z-5 because they were unable to excite any dentinal nerves with chemical stimuli. This discrepancy may have stemmed from the fact that the solution they used did not diffuse through the dentinal tubules to excite afferent nerves in the inner and predentinal layers. Thus, their techniques do not warrant statements about terminations. Some investigators studied nerve fibers with the light microscope but could not interpret exactly where the fibers terminatedF -lo Others used the electron microscope and described nerve terminations in the dentin that had cytologic characteristics identical to nerve terminations described in other locations. 1z-z8 The current work is consistent with the electron microscopic studies on nerve terminations. Only the nerve terminations were studied by using an accepted neuroanatomic method for locating such terminations.19-25 The rat incisor is a continually erupting tooth that shows continuously developing enamel and dentin at its 131
JOURNAL OF ENDODONTICS I VOt 3, NO 4, A n ' ~
apical inferior border3 8 Trigeminal nerve terminations were found in the predentin that would have become the middle dentin if the tooth had matured. Nerve terminations also were found in the subodontoblastic zone and the pulp of this immature tooth. The rat molar has been used by many investigators because of its similarity to the human molar. 28 In this study, no trigeminal nerve terminations were found in the pulp inferior to the subodontoblastic zone of the pulp horn. Th e subodontoblastic zone did contain trigeminal nerve endings; this is consistent with other studies. 15,18 The odontoblastic layer did not contain any trigeminal nerve terminations. Investigators who used the electron microscope have shown nerve axons to pass between odontoblasts, but were unable to show a direct contact between nerve fibers and odontoblasts. T M In addition, these experimenters found an attachment between nerve fibers and the odontoblastie process in the predentin and inner dentin but not in the outer dentin. In this study, trigeminal nerve terminations were noticed in these same areas. These terminations are functional nerve endings receiving a precursory protein from their cell body. This study raises several questions: Are the structures ascending in the layers one nerve with branches or terminations to each layer, or are they each a separate nerve to each layer? Are the subodontoblastic zone nerve terminations from myelinated nerve fibers and the dentinal nerves from unmyelinated nerves? Do the nerves attached to the odontoblastic process in dentin respond only to stimuli that affect the osmotic pressure, flow of fluid, a n d / o r membrane of the odontoblastic process? Do the nerve terminations in the subodontoblastic zone respond only to stimuli that change the intrapulpal pressure? Are there any efferent sympathetic nerve terminations in the dentin? 132
19~
o incisor Odontoblostic
0
pt m o l a r
n
2 M molar
A
3'd m o l a r
layer
3.00
P=.01 2.76
8 t. Q
2.00
4-.
1.00
0.00
A
@ o
O >
O
[]
z~
o
Graph 4--Odontobld layer did not show m significant t values in trigeminal ganglion.
O O
O
II o
O []
o D
o
2
3
4
Hours post injection o incisor Graph 5--Only developing apical inferior incisor showed significant t values at two, ]our, and five hours in pulp.
0
1*t m o l a r
a
2 nd m o l a r
A
3 'd m o l a r
Pu Ip 5.00
O
4.00
@ 9 3.00
P-I
2.7e A 13
2.00
o []
o
1.00
o o
o
3
4
0.00
2
Hoers post injection
5
Middle 5.00
dentin
o i ,t molar
[] A
[] 2 "d molar 4.00
[] A
A 3 'd molar
0
@ 0
3.00
Graph 6--1unction o f middle and inner dentin was termed middle dentin. Significant t values were noticed in /irst molar at two and three hours, all molars at [our hours, and second and third molars at five hours.
P=.OI
2.76
0
Predentin
@
6.00
2.00
Graph 8 In predentin, signi[icant t values were determined in incisor at two, four, and five hours, first molar a t / o u r hours, and second molar at five hours.
[] incisor
1.00
molar
0
pt
El
2 "d molar
L~
3 'd molar
5.0C
[]
O
4.0(3
0.00 2
3
4
Hours post injection
Q -:b
3.00
O
2.76
Inner dentin
7.00
[]
o incisor 6.00
O
I st
[] 2 "~ molar 5.00
A 3,d molar
A
o
2.00
B
Graph 7----Signi/icant t values were noticed in inner dentin of all molars on experimental side at ]our and five hours.
molar
P=.01
A
8
I .OC
r~
[]
[] A
0.0( 2 O
4.00
Z~
5
4
Hours post injection
[]
O
o incisor
A
3.00 2.76
3
[]
A 2.00
o I'~ molar
ra
@ @
P=.01
Subodontoblastic
o
zone
n 2"d molar
5.00
o
/, 3 'd molar []
o
o
1.00
A
O
4.00
o
0.00 2
3
4
Hours post inject ion
m Q
A
5.00 2.76
o
[]
P=.01
O Z~ o
ZOO
O
@ @
I.OO
Graph 9 Incisor at two hours and second and third molars a t / o u r and five hours showed significant t values in subodontoblastic zone.
0 0 @ n
0 0
o.oo
D
2
3
4
Hours p o s t injection
Summary
Reterences
A neuroanatomic method using the fast-axon transport of a radioactive isotope showed the termination of the trigeminal nerve in rat molars and incisors. Autoradiographic mean silver grain counts of experimental and control teeth were compared using Student's t tests. Statistically significant t values ( P = 0 . 0 1 ) showed that trigeminal nerve terminations were found in the middle dentin, inner dentin, predentin, and subodontoblastic zone, but not in the outer dentin, odontoblastic layer, or pulp of the rat molars. In the apical rat incisor, nerve terminations were found at the pulpodentinal membrane, subodontoblastic zone, and pulp, but not in the enamel, dentin, or odontoblastic layers.
1. Anderson, D.J.; Curwen, M.P.; and Howard, L.V. The sensitivity of human dentin. J Dent Res 37:669 Aug 1958. 2. Anderson, D.J., and Naylor, M.N. Chemical excitants of pain in human dentine and dental pulp. Arch Oral Biol 7:413 May-June 1962. 3. Anderson, D.J., and Matthews, B. An investigation into the reputed desensitizing effect of applying silver nitrate and strontium chloride to human dentine. Arch Oral Biol 11:1129 Nov 1966. 4. Dellow, P.G., and Roberts, M.L. Bradykinin application to dentine; a study of a sensory receptor mechanism. Aust Dent J 11:384 Dec 1966. 5. Brannstr6m, M., and Astr6m, A. A study on the mechanism of pain elicited from the dentin. J Dent Res 43:619 July-Aug 1964. 6. Van Hassel, H.J., and Ervin, M. Correlation of sensory response with intrapulpal temperature and pressure. J Dent Res 53:144 Feb 1973. 7. Held, A.J., and Baud, C.A. The innervation of the dental organ. Oral Surg 8:1262 Dec 1955. 8. Hattyasy, D. Continuous regeneration of the dentinal nerve-endings. Nature 189:72 Jan 7, 1961. 9. L6rfinth, C., and Csfinyi, K. Innervation of teeth. Acta Morph Acad Sci Hung 15:391, 1967. 10. Fearnhead, R.W. The histological demonstration of nerve fibres in human dentine. In Anderson, D.J., ed. Sensory mechanisms in dentine. New York, MacMillan Co., 1963, p 15. 11. Arwill, T. The ultrastructure of the pulpo-dentinal border zone. In Symons, N.B.B., ed. Dentine and pulp: their structure and reactions. London, Churchill Livingstone, 1968, p 147. 12. Frank, R.M. Etude au microscope electronique de l'odontoblaste et du canalicule dentinaire humain. Arch Oral Biol 11:179 Feb 1966. 13. Frank, R.M. Attachment sites between the odontoblast process and the intradentinal nerve fibre. Arch Oral Biol 13:833 July 1968. 14. Frank, R.M. Ultrastructural relationship between the odontoblast, its process and the nerve fibre. In Symons, N.B:B., ed. Dentine and pulp: their
*Hamilton Industries, Two Rivers, Wis. "~Eastman Kodak Co., Rochester, NY. This paper was presented at the annual meeting of the American Association of Endodontists on April 14, 1974, in San Diego, Calif; its authors were the recipients of the Endodontic Research Award. This article is derived from a thesis submitted by Dr. Menke to the faculty of the graduate school of Loyola University of Chicago in partial fulfillment of the requirements for the master of science degree. Dr. Menke is in private practice limited to endodontics in Worthington, Ohio. Dr. Weine is professor and director, postgraduate endodontics, Loyola University of Chicago, Maywood, IlL Dr. Ulinski is assistant professor, department of anatomy, University of Chicago. Dr. Smulson is professor and chairman, department of endodontics, Loyola University of Chicago. Requests for reprints should be directed to Dr. F. Weine, School of Dentistry, Loyola University of Chicago, 2160 S First Ave, Maywood, I11 60153.
134
structure and reactions. London, Churchill Livingstone, 1968, p 115. 15. Frank, R.M.; Sauvage, C.; and Frank, P. Morphological basis of dental sensitivity. Int Dent J 22:1 March 1972. 16. Johansen, E. Ultrastructure of dentine. In Miles, A.E.W., ed. Structural and chemical organization of teeth. New York, Academic Press Inc., 1967, vol 11, p 35. 17. Roane, J.B.; Foreman, D.W.; Melfi, R.C.; and Marshall, F.J. An ultrastructural study of dentinal innervation in the adult human tooth. Oral Surg 35:94 Jan 1973. 18. Corpron, R.E., and Avery, J.K. The ultrastructure of intradental nerves in developing mouse molars. Anat Rec 175:585 March 1973. 19. Droz, B., and Leblond, C.P. Axonal migration of proteins in the central nervous system and peripheral nerves as shown by radioautography. J Comp Neurol 121:325 Dec 1963. 20. Lasek, R. Protein transport in neurons. Int Rev Neurol 13:289, 1970. 21. Lasek, R. Axoplasmic transport in cat dorsal root ganglion cells: as studied with [3-H]-L-leucine. Brain Res 7:360 March 1968. 22. Sj6strand, J. Rapid axoplasmic transport of labelled proteins in the vagus and hypoglossal nerves of the rabbit. Exp Brain Res 8:105, 1969. 23. Schonback, J., and Cu6nod, M. Axoplasmic migration of protein. A light microscopic autoradiographic study in the avian retino-tectal pathway. Exp Brain Res 12:275, 1971. 24. Lasek, R.; Joseph, B.S.; and Whitlock, D.G. Evaluation of a radioautographic neuroanatomic tracing method. Brain Res 8:319 May 1968. 25. Cowan, W.M.; Gottlieb, D.I.; Hendrickson, A.E.; Price, J.L.; and Woolsey, T.A. The autoradiographic demonstration of axonal connections in the central nervous system. Brain Res 37:21 Feb 11, 1972. 26. Cariness, V.S., and Barkley, D.S. Section thickness and grain count variation in autoradiographic tritium. Stain Technol 46:131 May 1971. 27. Sawicki, W.; Blaton, O.; and Rowifiski, J. Correction of autoradiographic grain count in respect to precisely calculated background. Histochemie 26:67, 1971. 28. Schour, I., and Massler, M. The teeth. In Farris, E.J., ed. The rat in laboratory investigation. New York, Hafner Publishing Co., 1962, p 104.