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Response properties of periodontal mechanoreceptors in rats, in vitro
Brain Research Bulletin, Vol. 58, No. 4, pp. 357–361, 2002 Copyright © 2002 Elsevier Science Inc. All rights reserved. 0361-9230/02/$–see front matter
PII: S0361-9230(02)00771-2
Response properties of periodontal mechanoreceptors in rats, in vitro Noribumi Ishii,1 Kunimichi Soma1 and Kazuo Toda2,3∗ 1 Orthodontic
Science, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, Bunkyo-ku, Yushima, Tokyo, Japan; 2 Section of Cognitive Neurobiology, Department of Maxillofacial Biology, Graduate School, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan; and 3 Integrative Sensory Physiology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan [Received 20 June 2001; Revised 31 January 2002; Accepted 12 February 2002] the single sensory units. In the present study, we used an in vitro rat jaw-nerve preparation to stimulate the periodontal mechanoreceptors directly [31] and compared the characteristics of those receptors in the incisors and in the molars.
ABSTRACT: Single unit activities of the inferior alveolar nerve evoked by calibrated von Frey stimuli (1.1, 2.9, 7.8, 11.8, and 17.2 mN) on the periodontal ligaments of the mandibular molars or incisors were recorded in an in vitro jaw-nerve preparation of Wistar albino rats. The data of 55 (lower incisor) and 100 (lower molars) units were collected in the present study. Both rapidly (RA) and slowly adapting (SA) type units were found in the incisors, and most of these units were innervated by Aβ fibers. While all the units of the molars were of RA types, the innervated fibers of two-thirds (67/100) of the units have been identified as Aδ fibers. The response patterns of the RA type were subdivided into three types (ON, OFF or ON–OFF type) both in the incisors and the molars. While von Frey thresholds of all incisor units were 11.8 mN except one unit that was 7.8 mN, those of the molars varied from 2.9 to 11.8 mN. In the molars, a majority of afferents innervated the periodontal ligaments of more than one tooth. This study suggests that response properties of periodontal mechanoreceptors are different between the incisors and the molars in rats, suggesting that these receptors have different functions in the regulation of mastication. © 2002 Elsevier Science Inc. All rights reserved.
MATERIALS AND METHODS In Vitro Jaw-Nerve Preparation We used 63 female Wistar albino rats, weighing about 200 g, deeply anesthetized with thiamylal sodium (60 mg/kg, i.p., Isozol, Yoshitomi Pharmacy, Tokyo, Japan). We made an in vitro jaw-nerve preparation for the present study of which, detailed method has been published, were shown previously [31]. The mandible was divided into the right and left halves at the central suture by a pair of scissor. The major masticatory muscles (masseteric, temporal, medial pterygoid, lateral pterygoid, platysma, and digastric muscles) were detached from the bone. Then, the inferior alveolar nerve was identified at the foramen mandibulae and isolated from the surrounding tissue. The mandible on one side was removed together with the inferior alveolar nerve by cutting the temporomandibular joint. Thus, about 15–20 mm length of the inferior alveolar nerve trunk was obtained from the foramen mandibulae. The body temperature was maintained within the physiological range by means of a heating pad during the surgery. Disappearance of flexion and corneal reflexes were intermittently examined to confirm the depth of anesthesia, supplemental doses of thiamylal sodium was injected (15 mg/kg, i.p.), when necessary. The mandible was dipped into modified Krebs–Henseleit solution (110.9 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2 , 1.2 mM MgSO4 , 1.2 mM KH2 PO4 , 22.4 mM NaHCO3 , 20 mM glucose) that was saturated with 95% O2 –5% CO2 gas mixture and kept at 31◦ C using Peltier element. The direct stimulation of the incisal periodontal ligaments was facilitated by extraction of the incisors after cutting the lingual or the buccal side of the incisal alveolar bone using a dental drill. In case of the study for molar periodontal ligament, three molars of the mandible were extracted to expose the dental sockets and to stimulate the periodontal ligaments of the molars. We always confirmed the presence of
KEY WORDS: Periodontal ligament, Mechanoreceptor, von Frey hair, Jaw-nerve preparation, Rat.
INTRODUCTION Inputs from the periodontal sensory receptors are considered to be important information that control jaw movements [9,17]. Especially, those from the mechanoreceptors are critical for various trigeminal reflexes such as the jaw-opening reflex or the periodontal-masseter reflex, and also for masticatory control [7,30]. Therefore, clarification of the response properties of periodontal mechanoreceptors is thought to be important for better understanding of trigeminal motor functions. The majority of previous studies concerning periodontal mechanoreceptors have been done by application of stimuli on teeth in humans and/or various laboratory animals [1,8,14,17,25]. In these studies, it is difficult to clarify the physiological properties of periodontal afferents exactly. It is essential to apply mechanical stimuli directly to the periodontal ligament for the quantitative analysis of responses of
∗ Address for correspondence: Dr. Kazuo Toda, Division of Integrative Sensory Physiology, Department of Developmental and Reconstructive Medicine, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan. Fax: +81-95-849-7639; E-mail: [email protected]
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periodontal tissue remaining in the dental socket using a binocular microscope. Study Design The mandible was placed in the test pool and the inferior alveolar nerve was passed through a hole in the separating board to the recording chamber (oil pool). After ligating the central end of the inferior alveolar nerve with cotton thread, a moderate tension was given so as to insert the recording electrode easily. A tungsten microelectrode (tip impedance: 10–12 MΩ at 10 kHz) was inserted into the inferior alveolar nerve trunk using a micromanipulator (MP-1, Narishige, Tokyo), following the method of microneurography to record action potentials of the single nerve fibers [6,11,13]. The test pool was perfused with Krebs–Henseleit solution (0.3 ml/s, 31◦ C) during recording. Mechanical stimuli from various directions were applied to the periodontal ligament with 0.2 mm tip diameter von Frey hair (ranged: 1.1, 2.9, 7.8, 11.8, and 17.2 mN), and the threshold values of single mechanoreceptive units were determined. The unit that bursted 1 s or more after mechanical stimulation was classified as SA type. While the unit which bursted within 1 s was classified as RA type. ON type unit showed spikes only at the start of stimulation, OFF type unit only at the end of stimulation, and ON–OFF type unit both at the start and the end of stimulation. To measure conduction velocity of afferent fibers, electrical stimulation (duration: 0.1 ms; strength: 0.5–1 mA; frequency: 1 Hz) was applied to the inferior alveolar nerve trunk near the foramen mandibulae. The conduction velocity of each nerve fiber was estimated from the distance and the conduction time between the stimulating and recording electrodes, and corrected to the value at 37◦ C using the Q10 correlation reported by Paintal [24]. We classified the nerve fibers into three groups according to their conduction velocities, >10 m/s as Aβ fibers, 2–10 m/s as Aδ fibers, <2 m/s as C fibers according to the criteria of Kolzenburg et al. [15]. Statistics Mann–Whitney U-test was used to determine the statistical significance of the data from two groups and the Kruskal–Wallis or the Fisher’s PLSD post hoc test was used for comparison of data from more than three groups. RESULTS We recorded activities of 55 periodontal units in the incisor and 100 in the molars in the present study. The frequencies of the initial spontaneous activity in our jaw-nerve preparation were none in all units tested.
FIG. 1. Typical examples of the response of incisal periodontal units. (A) RA type: (a) stimulating site for 11.8 mN von Frey hair (∗); (b) responses of three stimulations. Bars under the trace indicate stimulus periods. (B) SA type: (a) stimulating site for 11.8 mN von Frey hair (∗); (b) response for continuous stimulation. Bar under the trace indicates stimulation period. Calibrations: 0.1 mV, 5 s.
Incisor Incisor units were divided into two types according to their response pattern. A rapidly adapting (RA) ty pe, and a slowly adapting (SA) type. SA type showed continuous spiking during von Frey stimulation (Fig. 1A and B, respectively). The RA type was further subclassified into ON type, OFF type and ON–OFF type groups. Unit types and innervating nerve fibers. Forty RA type units and 15 SA type units were obtained (Fig. 2A). Thirty-five ON type, one OFF type and four ON–OFF type units were observed within the RA type units. We did not observe changes in response pattern, even if the direction and the intensity of the stimulation were changed (Fig. 2B). These units were innervated by Aβ, Aδ, or C fibers. The conduction velocity of Aβ units were 26.4 ± 1.7 m/s (mean ± SEM, n = 38), Aδ units were 7.5 ± 0.5 m/s (n = 16) and C unit was 2.0 m/s (n = 1). Twenty-seven of RA and 11 of SA units were found within the Aβ innervated units, and 13 of RA and three of SA units were found within the Aδ innervated units. The C fiber innervated unit was found to be of the SA type (Table 1).
FIG. 2. Characteristics of incisal periodontal units. (A) Number of RA and SA type units recorded in incisal periodontal ligaments distinguished with unit types, respectively. (B) Number of units recorded in incisal periodontal ligaments according to their response patterns (ON, OFF, and ON–OFF type, respectively).
PERIODONTAL MECHANORECEPTORS
359 TABLE 1
CHARACTERISTICS OF PERIODONTAL MECHANORECEPTORS
Aβ (n = 38)
Fiber Unit type
Aδ (n = 16)
RA
RA
Response
OFF
ON
ON–OFF
CV Range von Frey Range n
15.30 11.80
28.55 ± 2.18 13.80–56.70 11.80
19.10 18.90–19.30 11.80
1
24
2
Fiber
SA Incisora 24.00 ± 2.99 12.20–44.40 11.44 ± 0.36 7.80–11.80 11
Aβ (n = 33)
Aδ (n = 67)
RA
RA
Unit type Response
ON
ON–OFF
ON
ON–OFF
CV Range von Frey Range n
13.34 ± 1.00 10.09–21.21 9.73 ± 0.83 2.90–11.80 12
Molara 19.10 ± 0.20 18.90–19.30 15.13 ± 1.23 2.90–11.80 21
7.76 ± 0.44 5.20–9.40 9.70 ± 0.57 2.90–11.80 28
9.20 ± 0.20 9.00–9.40 9.58 ± 0.44 2.90–11.80 39
a
C (n = 1)
ON
ON–OFF
SA
SA
7.76 ± 0.44 5.20–9.40 11.80
9.20 9.00–9.40 11.80
5.20 ± 1.97 2.80–9.10 11.80
2.00 11.80
11
2
3
1
CV, conduction velocity (m/s); von Frey, von Frey threshold (mN). CV and von Frey are represented as mean ± SEM.
FIG. 3. Typical examples of responses of periodontal units in the molars. (A) ON type: (a) stimulating site for 11.8 mN von Frey hair (II); (b) responses to stimulation. Bar under the trace indicates stimulation periods. (B) ON–OFF type: (a) stimulating site for 7.8 mN von Frey hair (II); (b) response for continuous stimulation. Bar under the trace indicates stimulation period. Calibrations: 0.1 mV, 5 s.
Threshold to mechanical stimulation. The threshold of one unit to the mechanical stimulation by von Frey hair was 7.8 mN and that of all others was 11.8 mN. There were no significant differences in the threshold values among the different unit types. Molar Typical examples of the molar periodontal units are shown in Fig. 3A and B; (A) was a record of ON type and (B) shows a record of ON–OFF type unit. Unit types and innervating nerve fibers. Forty ON type units and 60 ON–OFF type units were identified (Fig. 4). Units that changed the response pattern with the direction of the stimulus were not observed. Thirty-three units had conduction velocities in the Aβ range 14.5 ± 0.9 m/s (mean ± SEM, n = 33), Aδ range 6.7 ± 0.2 m/s (n = 67) (Table 1). Threshold to mechanical stimulation. Thresholds of mechanical stimulation with von Frey hair were 2.9 mN in one unit, which
FIG. 4. Histogram of the units recorded in molar periodontal ligaments distinguished by response patterns (ON and ON–OFF type, respectively).
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FIG. 5. Histogram of units recorded in molar periodontal ligaments according to the number of teeth whose periodontal mechanoreceptors were responding.
was innervated by Aβ fiber, 7.8 mN in 38 units and 11.8 mN in 53 units. A majority of molar units had a threshold lower than that of incisors. Thresholds of both Aβ and Aδ units were 2.9, 7.8 or 11.8 mN. The mean threshold value of Aβ units were 9.2 and 9.6 mN in Aδ innervated units. According to the unit type, the mean threshold value of ON type units was 9.7 mN (2.9–11.8 mN) and that of ON–OFF type units was 9.3 mN (2.9–11.8 mN). There was no significant difference between the two types (Mann–Whitney U-test: p > 0.05). Number of innervated teeth in the molar. We classified the units depending on how many molars were innervated by single fiber. The rat had three molars on one side of the mandible and these units were classified into three groups according to the number of teeth responding to mechanical stimulation as shown in Fig. 5. The number of units responding to mechanical stimulation of only one tooth was 24, those responding to two teeth were 23 and those responding to all three teeth were 53. In Aβ units, 6 responded to stimulation of only one tooth, 9 units responded to two different teeth and 18 units responded to all three teeth. Aδ innervated units that responded to only one tooth were 18, those responded to two teeth were 14 and those responded to all three teeth were 35. The threshold values of Aβ unit responding to only one tooth were 9.8 ± 0.9 mN (7.8–11.8 mN, n = 6), those responding to two teeth were 8.6 ± 1.0 mN (2.9–11.8 mN, n = 9), and those responding to three teeth were 9.3 ± 0.7 mN (2.9–11.8 mN, n = 18). In Aδ units, the threshold values of the units responding to one tooth were 10.9±0.4 mN (7.8–11.8 mN, n = 18), those responding to two teeth were 10.4 ± 0.5 mN (7.8–11.8 mN, n = 14), and those responding to three teeth were 8.7 ± 0.6 mN (2.9–11.8 mN, n = 35). DISCUSSION Incisor On the basis of response patterns to mechanical stimulation, two main types of periodontal units were found in the present study. One was SA type that was firing during continuous mechanical stimulation and the other was RA type that fired on either at the starting point, at the end point or at both points of the stimulation. The response pattern of the incisor units was classified into three types (ON, ON–OFF and OFF type). As stated before, a majority of units were of the ON type. In in vivo studies about the adaptation type of periodontal mechanoreceptors in cat, one study reported that there were two types of receptors of RA and SA type [4,20,21]. The other study
claimed that there was only one adaptation type and that the rate of adaptation was dependent on the location of the receptors within the periodontal tissues [18,19]. In rabbits, it was reported that all the receptors were of the RA type in the incisors [2]. In our in vitro study exposing the periodontal ligament, the mechanical stimulation was applied directly to the mechanoreceptors in the periodontal ligament. Therefore, we could stimulate periodontal mechanoreceptors with various intensities from various directions. From our results, we concluded that both RA and SA types exist in the periodontal ligaments, at least in the rat incisor. It is generally assumed that the fibers transmitting responses to non-noxious stimuli are Aβ fibers and the fibers transmitting responses to the noxious stimuli are Aδ or C fibers [5]. The majority (38/55, 69.1%) of the nerve fibers that transmitted the responses to non-noxious mechanical stimulation in the present study consisted of Aβ fibers. However, about 30% (16/55) of the innervating fibers were Aδ fibers. It is suggested that periodontal mechanoreceptors innervated by Aδ fibers may be similar to the mechanoreceptors (D-hair receptors) in the hairy skin, which have low threshold and are equally innervated by Aδ fibers [3,10,16,22,26]. The role of the rat mandibular incisors in mastication is considered to be the cutting up and crushing of foods. The pattern of jaw movements is reported to be as complex in rats as in humans [33,34]. Therefore, if mechanoreceptors of the incisor periodontal ligament can be aware of the time when the food is touched in the mouth, it is reasonable that these mechanoreceptors could play a role to start masticatory movements. In addition, the finding that the SA type was seen in the incisor periodontal ligament may be suggested to be useful to control bite force at the start of food intake. Molar The type of unit was quite different from the incisor. It was suggested that surgery could be influential in interfering with electrophysiological recording in our study. However, as described before, the frequencies of the initial spontaneous activity in our jaw-nerve preparation were almost none in the incisor and the molars, suggesting that, if any, the damages on the incisor and molar periodontal ligaments are similar. We found only the RA type in the rat molar and could not observe the SA type. Both Aδ (67%) and Aβ fibers (33%) were found in the molar periodontium. Two kinds of response patterns (ON type and ON–OFF type) were found in the RA type. The ON type was observed in 40%, while the ON–OFF type was found in 60% of the total. This difference seen in the molar may be related to the difference of the role in the jaw movements between the incisors and the molars. While the role of the incisors is to amputate and crush foods, that of the molars is to chew the foods [33,34]. Generally, in various species of animals, it is suggested that SA units play a role for detecting bite force during chewing [33,34]. However, in rats, ON–OFF RA units may play a role for detecting bite force instead of SA units, because RA type unit was predominant in the rat periodontal ligament. The threshold value of a majority of molar mechanoreceptors was lower than that of incisor mechanoreceptors. This would correspond to relatively smaller and soft foods crushed by molars, therefore, a lower threshold in molars than that in the incisors may be needed to detect food properties. Food is broken smaller by incisors and become soft by mixing with saliva in rat. This softened food is chewed by molars. Therefore, we suggest that the threshold value of molars is lower than that of incisors. This finding contrasts with earlier findings obtained from human data [23]. This discrepancy may be due to difference in animal species based on the difference in the food which human or rat is eating.
PERIODONTAL MECHANORECEPTORS It has been reported that the nerve fibers innervating periodontal mechanoreceptors branch off and had a receptive field among some teeth in the cat, showing that a nerve fiber can innervate the periodontal mechanoreceptors of more than one tooth [29]. In the rats, there were 76 units that showed responses from the periodontal ligament of more than one molar similar to the findings in cats [29]. Interestingly, one unit responded to the mechanical stimuli on periodontal ligaments in the first molar and the third molar. In the present study, all the molars were extracted before electrophysiological recordings. Therefore, mechanical stimulation could not be translated by mechanical coupling [12,27,28,32]. In conclusion, differences of the properties of the periodontal mechanoreceptors between incisors and molars in rat mandible were shown clearly in our in vitro study, suggesting that these receptors have different functions in the regulation of masticatory movements. ACKNOWLEDGEMENTS
We thank Dr. K.G. Imendra (Nagasaki University) for his valuable comments and discussions. This study was supported in part by a grant for Ministry of Education, Culture, Sports, Science and Technology of Japan (KT12671800; 2000, 2001). REFERENCES 1. Anderson, D. J.; Hannam, A. G.; Matthews, B. Sensory mechanisms in mammalian teeth and their supporting structures. Physiol. Rev. 336:15–16; 1970. 2. Appenteng, K.; Lund, J. P.; Séin, J. Intraoral mechanoreceptor activity during jaw movement in the anesthetized rabbit. J. Neurophysiol. 48:27–37; 1982. 3. Barasi, S.; Lynn, B. Effects of sympathetic stimulation on mechanoreceptive and nociceptive afferent units from the rabbit pinna. Brain Res. 378:21–27; 1986. 4. Cash, R. M.; Linden, R. W. The distribution of mechanoreceptors in the periodontal ligament of the mandibular canine tooth of the cat. J. Physiol. (London) 330:439–447; 1982. 5. Dubner, R.; Sessle, B. J.; Storey, A. T. The neural basis of oral and facial function. New York: Plenum Press; 1978. 6. Edwall, L.; Olgart, L. A new technique for recording of intradental sensory nerve activity in man. Pain 3:121–125; 1977. 7. Hannam, A. G. Reflex jaw opening in response to stimulation of periodontal mechanoreceptors in the cat. Arch. Oral Biol. 14:415–419; 1969. 8. Hannam, A. G. Receptor fields of periodontal mechanosensitive units in the dog. Arch. Oral Biol. 15:971–978; 1970. 9. Hannam, A. G. The innervation of the periodontal ligament. In: Berkovitz, B. K. B., ed. The periodontal ligament in health and disease. Oxford: Pergamon Press; 1982:173–196. 10. Horch, K. W. Ascending collaterals of cutaneous neurons in the fasciculus gracilis of the cat. Brain Res. 117:1–17; 1976. 11. Iwata, K.; Tsuboi, Y.; Toda, K.; Yagi, J.; Tujimoto, C.; Sumino, R. Comparisons of the sensation perceived and intradental nerve activity following temperature changes in human teeth. Exp. Brain Res. 87:213–217; 1991.
361 12. Jerge, C. R. Organization and function of the trigeminal mesencephalic nucleus. J. Neurophysiol. 26:379–392; 1963. 13. Johansson, R. S.; Olsson, K. Micro-electrode recordings from human oral-mechanoreceptors. Brain Res. 118:307–311; 1976. 14. Kieschke, J.; Mense, S. The use of a nerve-muscle preparation for studying nociceptors in vitro. Pain Suppl. 2:S6; 1984. 15. Kolzenburg, M.; Kress, M.; Reeh, P. W. The nociceptor sensitization by bradykinin dose not depend on sympathethic neurons. Neuroscience 46:465–473; 1992. 16. Leem, J. W.; Willis, W. D.; Chung, J. M. Cutaneous sensory receptors in the rat foot. J. Neurophysiol. 69:1684–1699; 1993. 17. Linden, R. W. Periodontal mechanoreceptors and their functions. In: Taylor, A., ed. Neurophysiology of the jaw and teeth. London: Macmillan; 1990:52–88. 18. Loescher, A. R.; Robinson, P. P. Receptor characteristics of periodontal mechanosensitive units supplying the cat’s lower canine. J. Neurophysiol. 62:971–978; 1989. 19. Loescher, A. R.; Robinson, P. P. Properties of periodontal mechanoreceptors supplying the cat’s lower canine at short and long periods after reinnervation. J. Physiol. 444:85–89; 1991. 20. Lund, J. P.; Millar, B. J. The response characteristics of mechanoreceptors related to their position in the cat canine periodontal ligament. Arch. Oral Biol. 33:51–56; 1988. 21. Lund, J. P.; Millar, B. J. The relation between the response properties and the position of reinnervated periodontal ligament mechanoreceptors in the cat. J. de Biol. Buccale 20:203–206; 1992. 22. Lynn, B.; Carpenter, S. E. Primary afferent units from the hairy skin of the rat hind limb. Brain Res. 238:29–43; 1982. 23. Manly, R. S.; Pfaffman, C.; Lathrop, D. D.; Keyser, J. Oral sensory thresholds of persons with natural and artificial dentitions. J. Dent. Res. 31:305–312; 1952. 24. Paintal, A. S. Effects of temperature on conduction in single vagal and saphenous myelinated nerve fibers of the cat. J. Physiol. (London) 180:20–49; 1965. 25. Pfaffmann, C. Afferent impulses from the teeth due to pressure and noxious stimulation. J. Physiol. (London) 97:207–219; 1939. 26. Ray, R. H.; Mallach, L. E.; Kruger, L. The response of single guard and down hair mechanoreceptors to moving air-jet stimulation. Brain Res. 346:333–347; 1985. 27. Robinsson, P. P. The course, relations and distribution of the inferior alveolar nerve and its branches in the cat. Anat. Rec. 195:265–272; 1979. 28. Sakada, S.; Kamio, E. Receptive fields and directional sensitivity of single sensory units innervating the periodontal ligaments of the cat mandibular teeth. Bull. Tokyo Dent. Coll. 12:25–43; 1971. 29. Tabata, T.; Watanabe, M.; Karita, K. Responses of somatosensory cortical neurones to tooth pressure and their modulation by transient mouth opening in the cat. Arch. Oral Biol. 31:735–740; 1986. 30. Tal, M. The threshold for eliciting the jaw opening reflex in rats is not increased by neonatal capsaicin. Behav. Brain Res. 13:197–200; 1984. 31. Toda, K.; Ishii, N.; Nakamura, Y. An in vitro jaw-nerve preparation for oral sensory study in the rat. J. Neurosci. Methods 61:85–90; 1995. 32. Trulsson, M. Multiple tooth receptive fields of single human periodontal mechanoreceptive afferents. J. Neurophysiol. 69:474–481; 1993. 33. Weijs, W. A.; Dantuma, R. Electromyography and mechanics of mastication in the albino rat. J. Morph. 146:1–33; 1975. 34. Weijs, W. A. Mandibular movements of the albino rat during feeding. J. Morph. 145:107–124; 1975.
PII: S0361-9230(02)00771-2
Response properties of periodontal mechanoreceptors in rats, in vitro Noribumi Ishii,1 Kunimichi Soma1 and Kazuo Toda2,3∗ 1 Orthodontic
Science, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, Bunkyo-ku, Yushima, Tokyo, Japan; 2 Section of Cognitive Neurobiology, Department of Maxillofacial Biology, Graduate School, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan; and 3 Integrative Sensory Physiology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan [Received 20 June 2001; Revised 31 January 2002; Accepted 12 February 2002] the single sensory units. In the present study, we used an in vitro rat jaw-nerve preparation to stimulate the periodontal mechanoreceptors directly [31] and compared the characteristics of those receptors in the incisors and in the molars.
ABSTRACT: Single unit activities of the inferior alveolar nerve evoked by calibrated von Frey stimuli (1.1, 2.9, 7.8, 11.8, and 17.2 mN) on the periodontal ligaments of the mandibular molars or incisors were recorded in an in vitro jaw-nerve preparation of Wistar albino rats. The data of 55 (lower incisor) and 100 (lower molars) units were collected in the present study. Both rapidly (RA) and slowly adapting (SA) type units were found in the incisors, and most of these units were innervated by Aβ fibers. While all the units of the molars were of RA types, the innervated fibers of two-thirds (67/100) of the units have been identified as Aδ fibers. The response patterns of the RA type were subdivided into three types (ON, OFF or ON–OFF type) both in the incisors and the molars. While von Frey thresholds of all incisor units were 11.8 mN except one unit that was 7.8 mN, those of the molars varied from 2.9 to 11.8 mN. In the molars, a majority of afferents innervated the periodontal ligaments of more than one tooth. This study suggests that response properties of periodontal mechanoreceptors are different between the incisors and the molars in rats, suggesting that these receptors have different functions in the regulation of mastication. © 2002 Elsevier Science Inc. All rights reserved.
MATERIALS AND METHODS In Vitro Jaw-Nerve Preparation We used 63 female Wistar albino rats, weighing about 200 g, deeply anesthetized with thiamylal sodium (60 mg/kg, i.p., Isozol, Yoshitomi Pharmacy, Tokyo, Japan). We made an in vitro jaw-nerve preparation for the present study of which, detailed method has been published, were shown previously [31]. The mandible was divided into the right and left halves at the central suture by a pair of scissor. The major masticatory muscles (masseteric, temporal, medial pterygoid, lateral pterygoid, platysma, and digastric muscles) were detached from the bone. Then, the inferior alveolar nerve was identified at the foramen mandibulae and isolated from the surrounding tissue. The mandible on one side was removed together with the inferior alveolar nerve by cutting the temporomandibular joint. Thus, about 15–20 mm length of the inferior alveolar nerve trunk was obtained from the foramen mandibulae. The body temperature was maintained within the physiological range by means of a heating pad during the surgery. Disappearance of flexion and corneal reflexes were intermittently examined to confirm the depth of anesthesia, supplemental doses of thiamylal sodium was injected (15 mg/kg, i.p.), when necessary. The mandible was dipped into modified Krebs–Henseleit solution (110.9 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2 , 1.2 mM MgSO4 , 1.2 mM KH2 PO4 , 22.4 mM NaHCO3 , 20 mM glucose) that was saturated with 95% O2 –5% CO2 gas mixture and kept at 31◦ C using Peltier element. The direct stimulation of the incisal periodontal ligaments was facilitated by extraction of the incisors after cutting the lingual or the buccal side of the incisal alveolar bone using a dental drill. In case of the study for molar periodontal ligament, three molars of the mandible were extracted to expose the dental sockets and to stimulate the periodontal ligaments of the molars. We always confirmed the presence of
KEY WORDS: Periodontal ligament, Mechanoreceptor, von Frey hair, Jaw-nerve preparation, Rat.
INTRODUCTION Inputs from the periodontal sensory receptors are considered to be important information that control jaw movements [9,17]. Especially, those from the mechanoreceptors are critical for various trigeminal reflexes such as the jaw-opening reflex or the periodontal-masseter reflex, and also for masticatory control [7,30]. Therefore, clarification of the response properties of periodontal mechanoreceptors is thought to be important for better understanding of trigeminal motor functions. The majority of previous studies concerning periodontal mechanoreceptors have been done by application of stimuli on teeth in humans and/or various laboratory animals [1,8,14,17,25]. In these studies, it is difficult to clarify the physiological properties of periodontal afferents exactly. It is essential to apply mechanical stimuli directly to the periodontal ligament for the quantitative analysis of responses of
∗ Address for correspondence: Dr. Kazuo Toda, Division of Integrative Sensory Physiology, Department of Developmental and Reconstructive Medicine, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan. Fax: +81-95-849-7639; E-mail: [email protected]
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periodontal tissue remaining in the dental socket using a binocular microscope. Study Design The mandible was placed in the test pool and the inferior alveolar nerve was passed through a hole in the separating board to the recording chamber (oil pool). After ligating the central end of the inferior alveolar nerve with cotton thread, a moderate tension was given so as to insert the recording electrode easily. A tungsten microelectrode (tip impedance: 10–12 MΩ at 10 kHz) was inserted into the inferior alveolar nerve trunk using a micromanipulator (MP-1, Narishige, Tokyo), following the method of microneurography to record action potentials of the single nerve fibers [6,11,13]. The test pool was perfused with Krebs–Henseleit solution (0.3 ml/s, 31◦ C) during recording. Mechanical stimuli from various directions were applied to the periodontal ligament with 0.2 mm tip diameter von Frey hair (ranged: 1.1, 2.9, 7.8, 11.8, and 17.2 mN), and the threshold values of single mechanoreceptive units were determined. The unit that bursted 1 s or more after mechanical stimulation was classified as SA type. While the unit which bursted within 1 s was classified as RA type. ON type unit showed spikes only at the start of stimulation, OFF type unit only at the end of stimulation, and ON–OFF type unit both at the start and the end of stimulation. To measure conduction velocity of afferent fibers, electrical stimulation (duration: 0.1 ms; strength: 0.5–1 mA; frequency: 1 Hz) was applied to the inferior alveolar nerve trunk near the foramen mandibulae. The conduction velocity of each nerve fiber was estimated from the distance and the conduction time between the stimulating and recording electrodes, and corrected to the value at 37◦ C using the Q10 correlation reported by Paintal [24]. We classified the nerve fibers into three groups according to their conduction velocities, >10 m/s as Aβ fibers, 2–10 m/s as Aδ fibers, <2 m/s as C fibers according to the criteria of Kolzenburg et al. [15]. Statistics Mann–Whitney U-test was used to determine the statistical significance of the data from two groups and the Kruskal–Wallis or the Fisher’s PLSD post hoc test was used for comparison of data from more than three groups. RESULTS We recorded activities of 55 periodontal units in the incisor and 100 in the molars in the present study. The frequencies of the initial spontaneous activity in our jaw-nerve preparation were none in all units tested.
FIG. 1. Typical examples of the response of incisal periodontal units. (A) RA type: (a) stimulating site for 11.8 mN von Frey hair (∗); (b) responses of three stimulations. Bars under the trace indicate stimulus periods. (B) SA type: (a) stimulating site for 11.8 mN von Frey hair (∗); (b) response for continuous stimulation. Bar under the trace indicates stimulation period. Calibrations: 0.1 mV, 5 s.
Incisor Incisor units were divided into two types according to their response pattern. A rapidly adapting (RA) ty pe, and a slowly adapting (SA) type. SA type showed continuous spiking during von Frey stimulation (Fig. 1A and B, respectively). The RA type was further subclassified into ON type, OFF type and ON–OFF type groups. Unit types and innervating nerve fibers. Forty RA type units and 15 SA type units were obtained (Fig. 2A). Thirty-five ON type, one OFF type and four ON–OFF type units were observed within the RA type units. We did not observe changes in response pattern, even if the direction and the intensity of the stimulation were changed (Fig. 2B). These units were innervated by Aβ, Aδ, or C fibers. The conduction velocity of Aβ units were 26.4 ± 1.7 m/s (mean ± SEM, n = 38), Aδ units were 7.5 ± 0.5 m/s (n = 16) and C unit was 2.0 m/s (n = 1). Twenty-seven of RA and 11 of SA units were found within the Aβ innervated units, and 13 of RA and three of SA units were found within the Aδ innervated units. The C fiber innervated unit was found to be of the SA type (Table 1).
FIG. 2. Characteristics of incisal periodontal units. (A) Number of RA and SA type units recorded in incisal periodontal ligaments distinguished with unit types, respectively. (B) Number of units recorded in incisal periodontal ligaments according to their response patterns (ON, OFF, and ON–OFF type, respectively).
PERIODONTAL MECHANORECEPTORS
359 TABLE 1
CHARACTERISTICS OF PERIODONTAL MECHANORECEPTORS
Aβ (n = 38)
Fiber Unit type
Aδ (n = 16)
RA
RA
Response
OFF
ON
ON–OFF
CV Range von Frey Range n
15.30 11.80
28.55 ± 2.18 13.80–56.70 11.80
19.10 18.90–19.30 11.80
1
24
2
Fiber
SA Incisora 24.00 ± 2.99 12.20–44.40 11.44 ± 0.36 7.80–11.80 11
Aβ (n = 33)
Aδ (n = 67)
RA
RA
Unit type Response
ON
ON–OFF
ON
ON–OFF
CV Range von Frey Range n
13.34 ± 1.00 10.09–21.21 9.73 ± 0.83 2.90–11.80 12
Molara 19.10 ± 0.20 18.90–19.30 15.13 ± 1.23 2.90–11.80 21
7.76 ± 0.44 5.20–9.40 9.70 ± 0.57 2.90–11.80 28
9.20 ± 0.20 9.00–9.40 9.58 ± 0.44 2.90–11.80 39
a
C (n = 1)
ON
ON–OFF
SA
SA
7.76 ± 0.44 5.20–9.40 11.80
9.20 9.00–9.40 11.80
5.20 ± 1.97 2.80–9.10 11.80
2.00 11.80
11
2
3
1
CV, conduction velocity (m/s); von Frey, von Frey threshold (mN). CV and von Frey are represented as mean ± SEM.
FIG. 3. Typical examples of responses of periodontal units in the molars. (A) ON type: (a) stimulating site for 11.8 mN von Frey hair (II); (b) responses to stimulation. Bar under the trace indicates stimulation periods. (B) ON–OFF type: (a) stimulating site for 7.8 mN von Frey hair (II); (b) response for continuous stimulation. Bar under the trace indicates stimulation period. Calibrations: 0.1 mV, 5 s.
Threshold to mechanical stimulation. The threshold of one unit to the mechanical stimulation by von Frey hair was 7.8 mN and that of all others was 11.8 mN. There were no significant differences in the threshold values among the different unit types. Molar Typical examples of the molar periodontal units are shown in Fig. 3A and B; (A) was a record of ON type and (B) shows a record of ON–OFF type unit. Unit types and innervating nerve fibers. Forty ON type units and 60 ON–OFF type units were identified (Fig. 4). Units that changed the response pattern with the direction of the stimulus were not observed. Thirty-three units had conduction velocities in the Aβ range 14.5 ± 0.9 m/s (mean ± SEM, n = 33), Aδ range 6.7 ± 0.2 m/s (n = 67) (Table 1). Threshold to mechanical stimulation. Thresholds of mechanical stimulation with von Frey hair were 2.9 mN in one unit, which
FIG. 4. Histogram of the units recorded in molar periodontal ligaments distinguished by response patterns (ON and ON–OFF type, respectively).
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FIG. 5. Histogram of units recorded in molar periodontal ligaments according to the number of teeth whose periodontal mechanoreceptors were responding.
was innervated by Aβ fiber, 7.8 mN in 38 units and 11.8 mN in 53 units. A majority of molar units had a threshold lower than that of incisors. Thresholds of both Aβ and Aδ units were 2.9, 7.8 or 11.8 mN. The mean threshold value of Aβ units were 9.2 and 9.6 mN in Aδ innervated units. According to the unit type, the mean threshold value of ON type units was 9.7 mN (2.9–11.8 mN) and that of ON–OFF type units was 9.3 mN (2.9–11.8 mN). There was no significant difference between the two types (Mann–Whitney U-test: p > 0.05). Number of innervated teeth in the molar. We classified the units depending on how many molars were innervated by single fiber. The rat had three molars on one side of the mandible and these units were classified into three groups according to the number of teeth responding to mechanical stimulation as shown in Fig. 5. The number of units responding to mechanical stimulation of only one tooth was 24, those responding to two teeth were 23 and those responding to all three teeth were 53. In Aβ units, 6 responded to stimulation of only one tooth, 9 units responded to two different teeth and 18 units responded to all three teeth. Aδ innervated units that responded to only one tooth were 18, those responded to two teeth were 14 and those responded to all three teeth were 35. The threshold values of Aβ unit responding to only one tooth were 9.8 ± 0.9 mN (7.8–11.8 mN, n = 6), those responding to two teeth were 8.6 ± 1.0 mN (2.9–11.8 mN, n = 9), and those responding to three teeth were 9.3 ± 0.7 mN (2.9–11.8 mN, n = 18). In Aδ units, the threshold values of the units responding to one tooth were 10.9±0.4 mN (7.8–11.8 mN, n = 18), those responding to two teeth were 10.4 ± 0.5 mN (7.8–11.8 mN, n = 14), and those responding to three teeth were 8.7 ± 0.6 mN (2.9–11.8 mN, n = 35). DISCUSSION Incisor On the basis of response patterns to mechanical stimulation, two main types of periodontal units were found in the present study. One was SA type that was firing during continuous mechanical stimulation and the other was RA type that fired on either at the starting point, at the end point or at both points of the stimulation. The response pattern of the incisor units was classified into three types (ON, ON–OFF and OFF type). As stated before, a majority of units were of the ON type. In in vivo studies about the adaptation type of periodontal mechanoreceptors in cat, one study reported that there were two types of receptors of RA and SA type [4,20,21]. The other study
claimed that there was only one adaptation type and that the rate of adaptation was dependent on the location of the receptors within the periodontal tissues [18,19]. In rabbits, it was reported that all the receptors were of the RA type in the incisors [2]. In our in vitro study exposing the periodontal ligament, the mechanical stimulation was applied directly to the mechanoreceptors in the periodontal ligament. Therefore, we could stimulate periodontal mechanoreceptors with various intensities from various directions. From our results, we concluded that both RA and SA types exist in the periodontal ligaments, at least in the rat incisor. It is generally assumed that the fibers transmitting responses to non-noxious stimuli are Aβ fibers and the fibers transmitting responses to the noxious stimuli are Aδ or C fibers [5]. The majority (38/55, 69.1%) of the nerve fibers that transmitted the responses to non-noxious mechanical stimulation in the present study consisted of Aβ fibers. However, about 30% (16/55) of the innervating fibers were Aδ fibers. It is suggested that periodontal mechanoreceptors innervated by Aδ fibers may be similar to the mechanoreceptors (D-hair receptors) in the hairy skin, which have low threshold and are equally innervated by Aδ fibers [3,10,16,22,26]. The role of the rat mandibular incisors in mastication is considered to be the cutting up and crushing of foods. The pattern of jaw movements is reported to be as complex in rats as in humans [33,34]. Therefore, if mechanoreceptors of the incisor periodontal ligament can be aware of the time when the food is touched in the mouth, it is reasonable that these mechanoreceptors could play a role to start masticatory movements. In addition, the finding that the SA type was seen in the incisor periodontal ligament may be suggested to be useful to control bite force at the start of food intake. Molar The type of unit was quite different from the incisor. It was suggested that surgery could be influential in interfering with electrophysiological recording in our study. However, as described before, the frequencies of the initial spontaneous activity in our jaw-nerve preparation were almost none in the incisor and the molars, suggesting that, if any, the damages on the incisor and molar periodontal ligaments are similar. We found only the RA type in the rat molar and could not observe the SA type. Both Aδ (67%) and Aβ fibers (33%) were found in the molar periodontium. Two kinds of response patterns (ON type and ON–OFF type) were found in the RA type. The ON type was observed in 40%, while the ON–OFF type was found in 60% of the total. This difference seen in the molar may be related to the difference of the role in the jaw movements between the incisors and the molars. While the role of the incisors is to amputate and crush foods, that of the molars is to chew the foods [33,34]. Generally, in various species of animals, it is suggested that SA units play a role for detecting bite force during chewing [33,34]. However, in rats, ON–OFF RA units may play a role for detecting bite force instead of SA units, because RA type unit was predominant in the rat periodontal ligament. The threshold value of a majority of molar mechanoreceptors was lower than that of incisor mechanoreceptors. This would correspond to relatively smaller and soft foods crushed by molars, therefore, a lower threshold in molars than that in the incisors may be needed to detect food properties. Food is broken smaller by incisors and become soft by mixing with saliva in rat. This softened food is chewed by molars. Therefore, we suggest that the threshold value of molars is lower than that of incisors. This finding contrasts with earlier findings obtained from human data [23]. This discrepancy may be due to difference in animal species based on the difference in the food which human or rat is eating.
PERIODONTAL MECHANORECEPTORS It has been reported that the nerve fibers innervating periodontal mechanoreceptors branch off and had a receptive field among some teeth in the cat, showing that a nerve fiber can innervate the periodontal mechanoreceptors of more than one tooth [29]. In the rats, there were 76 units that showed responses from the periodontal ligament of more than one molar similar to the findings in cats [29]. Interestingly, one unit responded to the mechanical stimuli on periodontal ligaments in the first molar and the third molar. In the present study, all the molars were extracted before electrophysiological recordings. Therefore, mechanical stimulation could not be translated by mechanical coupling [12,27,28,32]. In conclusion, differences of the properties of the periodontal mechanoreceptors between incisors and molars in rat mandible were shown clearly in our in vitro study, suggesting that these receptors have different functions in the regulation of masticatory movements. ACKNOWLEDGEMENTS
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