THE ANATOMICAL RECORD 234:144-152 (1992)
Anatomy and Developmental Chronology of the Rat Inferior Alveolar Nerve CARINA S. JOHANSSON, CLAES HILDEBRAND, AND BO POVLSEN Departments of Cell Biology (C.S.J.,C.H.) and Hand and Plastic Surgery (B.P.), Faculty of Health Sciences, University of Linkoping, 581 85 Linkoping, Sweden
ABSTRACT
This report describes the anatomy of the inferior alveolar neurovascular bundle in the adult rat and provides a quantitative analysis of the developing inferior alveolar nerve (IAN). Soon after its entrance in the mandibular canal, the IAN splits into a mental nerve (MN) and a n inferior dental nerve (IDN), which course in separate bony compartments. The MN passes unbranched through the mandibular canal. The IDN sends branches to the incisor, the first molar, and the second molar. The third molar (M,) is supplied by a separate IAN branch. The adult rat IAN contains 8,000-10,000 axons, 70%of which are myelinated. The MN accounts for 70%of all IAN axons, the IDN 26%,and 4%form the M, branch. The proportion of large myelinated axons is lower in the MN than in the IDN. Following chemical sympathectomy, the IAN axon number does not change in a statistically significant way. The total number of IAN axons, which is high prenatally and neonatally, has decreased to the adult level about 1 week after birth. De novo myelination commences at birth and is complete 3-4 weeks later. The size spectrum of the myelinated fibres is narrow and unimodal during the first postnatal weeks. By 1 month, the largest fibres reach diameters of -6 pm, and a bimodal pattern is emerging. From 3 months and on, the size range reaches up to 10-12 pm, and the distribution is bimodal. These data provide a basis for further studies on developmental tooth-nerve interactions. o 1992 Wiley-Liss, Inc.
Vertebrate teeth have a rich sensory innervation, and the dental pulp is a well-defined target structure, supplied by a fairly homogeneous nerve fibre population. From a morphogenetic point of view, the trigeminal branches projecting to the teeth are of particular interest, because of the ontogenetically late establishment of dental target contacts, the extensive developmental remodelling of the dentition in diphyodont and polyphyodont species, and the progressive tooth alterations during ageing (Miles, 1961; Fried and Hildebrand, 1981a,b; Mohamed and Atkinson, 1983, Fried, 1987). Much experimental work on tooth-nerve relations has been performed in the cat (Arwill e t al., 1973; Karlsson et al., 1974; Olgart et al., 1977; Robinson, 1979, 1981; Arvidsson and Gobel, 1981). Some studies describe tooth-nerve relations in the rat or the mouse (Byers and Holland, 1977;Pearson, 1977; Bishop, 1981; Mohamed and Atkinson, 1983; see Byers, 1984, 1985). Few reports deal with the basic biological mechanisms involved in the establishment and maintenance of tooth nerves. For such studies, the use of developing cellular explants in vitro might represent a rewarding approach (cf Lumsden and Davies, 1983, 1986; Naftel, 1987). From various points of view, the rat is more suitable than the cat for in vitro experiments on explanted tissues. Since the mandibular dentition is supplied by a single nerve trunk-the inferior alveolar nerve (IAN) (Greene, 1968; Hebel and Stromberg, 1986; Robinson, 1979)-it appears suitable for experimental studies. The anatomy and development of the feline IAN is elucidated in several studies (Robinson, c
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1979; Fried and Hildebrand, 1982a,b; Fried et al., 1982), but the rat IAN is less well known. The few anatomical descriptions available are rather incomplete (Ridehalgh and Stewart, 1938; Rehak, 1963). Apart from a recent report from our laboratory (Hildebrand and Johansson, 1991), we are aware of only one paper that deals with the developmental maturation of this nerve (Sugimoto et al., 1983). Morphological and maturational data on the rat IAN are needed as a basis for studies on developmental tooth-nerve interactions. Hence, the aim of the present work is to describe the anatomy of the inferior alveolar neurovascular bundle in the adult rat and to provide a chronology for the structural development of the rat IAN. Some of these results have been reported in abstract form (Johansson and Hildebrand, 1990). MATERIALS AND METHODS Dissection
Adult male and female Wistar and Sprague-Dawley rats were anaesthetized with ether and exsanguinated (n = 20). The mandible was removed, split in halves, and cleaned from soft tissues so that the mandibular and mental foramina and their contents became visible. The inferior alveolar neurovascular bundle was exposed by opening the mandibular canal from the buccal side, using a dental bur and fine rongeurs. The
Received September 12, 1991; accepted December 14, 1991.
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contents of the bundle were subjected to some blunt dissection and were examined in a Nikon SMZ-2T dissection microscope equipped with a Nikon F-301 camera. Immersion of the dissected specimen in saline simplified identification of small branches. Chemical Sympathectomy
Rat pups, of both sexes, were injected under the dorsal skin with a 1.5% solution of guanethidine sulphate (Sigma) in sterile water (50 mg/kg, pH adjusted to 7.4), 6 days per week for 3 weeks, beginning 3 days after birth (n = 4). This treatment destroys most neurons in the sympathetic ganglia (Chad et al., 1983, Johnson and Manning, 1984). These animals were allowed to survive for 3-6 months. At that age, they were perfused and the IAN was collected and prepared for electron microscopic examination as described below. Nerves from untreated age-matched animals used for the developmental part of this study (vide infra) served as controls (n = 6). Light Microscopy (LM)
To reach a satisfactory description of the various thin branches included in the inferior alveolar neurovascular bundle, it was necessary to perform an LM analysis of serial sections. For this purpose, adult rats were anaesthetized with chloral hydrate (30 mg/100 g i.p.) and perfused with glutaraldehyde, as described below (n = 5). After perfusion, the mandible was removed, postfixed, and rinsed in cold buffer. It was split in halves and cleaned from soft tissues. The crowns of the molars and part of the alveolar process were removed. Specimens composed of the portion of the mandibular corpus that contains the mandibular canal were demineralized in 4% EDTA (Sigma) in cacodylate buffer (pH 7.2-7.4,4" C, 6-8 weeks) (Warshawsky and Moore, 1967). The EDTA solution was changed three times weekly. The demineralized specimens were dehydrated in alcohol and embedded in JB4 resin (Bio-Rad).Transverse serial sections (3 Fm) were cut from the mandibular canal region with an LKB Historange microtome, using glass knives. Sections with a spacing of 0.1 mm were mounted in sequential order on glass slides, stained with toluidine blue, and examined in a light microscope. Drawings showing the different branches a t selected levels were made with the aid of a drawing tube. Electron Microscopy (EM)
Material intended for EM was collected from 13 rat fetuses aged fetal day 17-21 and from 38 postnatal rats aged from newborn to 30 months. Fetuses and young rat pups (0-5 days) were anesthetized by hypothermia. Older pups and adult rats were anesthetized with chloral hydrate. The rats were perfused with Tyrode's solution, followed by 5% glutaraldehyde dissolved in a 300 mOsm phosphate buffer, also containing 0.1 M sucrose. Specimens were taken from the IAN at its entrance in the mandibular canal, excluding the mylohyoid branch. The specimens were postfixed in 5% phosphate-buffered glutaraldehyde, rinsed in the buffer, osmicated in 2% buffered OsO,, and rinsed. After dehydration in acetone, the specimens were embedded in Vestopal W. Semithin toluidine blue-stained cross sections from the IAN were examined in the light
microscope for orientation. Thin cross sections were cut on an LKB Ultrotome I11 or IV, using glass or diamond knives. The sections were collected on one-hole copper grids coated with formvar. The thin sections were photographed in a Jeol 1200EX electron microscope. When dealing with the completely unmyelinated fetal nerves, all axons present in approximately one-half the cross-cut nerve were counted. After dividing the total area of the cross-cut nerve into squares, with the size of one microscopic field, every second square was photographed at x 15,000. The axons on the micrographs were counted, and the areas they occupied were measured with a Videoplan equipment (Kontron; Zeiss). This rocedure resulted in an average number of axons/Fm . By measuring the total area of the cross-cut nerve, the total number of axons could be estimated. With respect to nerves from rat pups younger than 4 days old, a t which time few myelinated axons have formed, every fourth square was processed as described above, and the total number of axons was determined in the same way. In nerves from older animals, the number of myelinated axons was obtained from montages of electron micrographs covering the entire nerve ( x 4,000-6,000). For determination of unmyelinated axon numbers, the sections were reexamined in the EM and all unmyelinated axons surrounding each of some 1,000 myelinated axons were counted. In each nerve, a t least five different areas, together covering approximately one-eighth of the total nerve area, were used for counting. The cross-sectional areas of - 1,000 myelinated fibres (myelin sheaths included) were measured on EM prints ( x 2,000-4,000) showing IAN cross sections from eight different postnatal ages (one animal/age), using a Videoplan equipment (Kontron; Zeiss). Fibre diameters were calculated from area values, assuming that they correspond to circle diameters (Karnes et al., 1977). In addition to countings and measurements on the adult IAN as a whole, the dental and mental branches, and the specific branch to the third molar were considered separately in three cases. These data were obtained from within 1mm distal to the bifurcation of the IAN into the mental nerve and the inferior dental nerve.
f
RESULTS Contents of the Mandibular Canal
The IAN courses along the lingual aspect of the mandibular ramus and emits the mylohyoid branch some 8 mm before the mandibular foramen, which it enters as a single coherent branch (Fig. 1).One artery and some small veins accompany the nerve, together forming the inferior alveolar neurovascular bundle (Fig. 3a). We could not see any separate bundles of small myelinated and unmyelinated axons in association with the blood vessels. Shortly after its entrance in the mandibular canal, the IAN emits a slender branch, which splits into three fascicles for the roots of the third molar (M3) (Figs. 1, 2, 3a). Below M,, the IAN splits into its two major branches, the mental nerve (MN) and the inferior dental nerve (IDN), the former being thicker than the latter (Fig. 1). Shortly after this nerve division, the mandibular canal bifurcates into a buccal portion, containing the MN, and a lingual portion, containing the
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well as in the young and old adult stages, axon number varies between 8,000 and 10,000 (Fig. 5). Separate counts in the MN and the IDN of adult rats showed that the average MN contains 6,250 axons (70%of all IAN axons), while the average IDN contains 2,630 axons (30%). The latter figure includes the branch to M,, which consists of 348 axons (4%). In the rats subjected to chemical sympathectomy, the average total number of axons was found to be 7,400 (k890; SD). The six age-matched control nerves contain 8,800 axons (5460; SD). Statistical analysis through the Wilcoxon rank-sum test showed that there is no significant difference between the guanethidine treated rats and the control group. Proportion of Myelinated Axons
Fig. 1. Photograph showing a dissected rat inferior alveolar nerve (IAN) and some of its branches: a, IAN nerve trunk; b, mental nerve; c, branch to the third molar; d, branch to the odontogenic root zone of the incisor; e, branch to the second molar; f, branch to the midportion of the incisal periodontium; g, branches to the first molar. X 30.
IDN (Fig. 3b). The bony septum that separates the two portions begins at a level between the M, and the second molar (M,). The MN extends unbranched through the buccal portion of the canal, exits through the mental foramen, and sends branches to the mucosa and skin of the lower lip and to the skin of the chin. The cutaneous branches cover an area of about 25 mm'. The IDN courses rostrally towards and into the lingual compartment of the mandibular canal, emitting a series of branches (Fig. 2). Shortly after its appearance as a separate nerve trunk, a thin recurrent loop leaves the IDN and courses to the odontogenic root zone of the incisor (Figs. 1, 2,3a). This twig, which is composed of a majority of unmyelinated axons and some four or five myelinated ones, is so thin that it can barely be discerned a t dissection. Below the M,, a branch with two fascicles courses rostrally towards the periodontium and the root apices of M, (Figs. 2, 3b). In the average rat, a tiny ventral branch composed of a few fascicles leaves the IDN below M, and enters the periodontium of the incisor (Fig. 2). In the individual case, the origin of this branch varies from below M, to below the first molar (MI). The small nerve fascicles to the periodontium of the incisor are composed of comparatively large myelinated axons (diameter = 8-9 pm) and a few unmyelinated axon bundles. The terminal branches of the IDN include two large and several small ramifications to the MI. A minor twig from the IDN courses to the most rostra1 periodontium of the incisor (Figs. 2, 3c). Number of Axons
Representative examples of the EM appearance of the IAN at some developmental stages are shown in Figure 4. In the youngest fetal rats examined (fetal day 17), the average IAN contains about 13,700 axons (range 11,400-16,500; Fig. 5). Subsequently, the number decreases, reaching a mean level of -11,100 at birth. The decrease has ceased 1 week postnatally, when the average number is about 8,800 (range 8,0009,400; Fig. 5). During the continued development, as
Throughout prenatal development, the IAN is composed of unmyelinated axons (Fig. 5).Myelination commences on the day of birth. Subsequently, the proportion of myelinated axons increases very rapidly, particularly during the second week (Fig. 5). One week postnatally this proportion is -15%, and by 10 days about one half of all axons have achieved myelin sheaths. From 3-4 weeks on, the proportion of myelinated axons varies around 70% (Fig. 5). Separate counts show that the proportion of myelinated axons is 65% in the MN and 81% in the IDN (including the M, branch). The separate branch to M, contains 67% myelinated axons. In chemically sympathectomized animals, the proportion of myelinated IAN axons is 69%, i.e., similar to that in normal nerves. Size Distribution of Myelinated Fibres
One week after birth, the myelinated IAN fibres exhibit a unimodal size spectrum with a diameter (D) range of 0.8-3 pm (Fig. 6). During the second week, the upper end of the size spectrum shifts to -4 pm. After 3 weeks, the maximum diameter is -6 pm, and there is a single peak a t 3 pm (Fig. 6). By 1 month, the size range extends from 1 to 6 pm, and the pattern is weakly bimodal. At about 2 months, D ranges from 1to 9 pm, and the pattern is bimodal (Fig. 6). In adult rats aged 3-12 months, the diameter range extends from 1 to 10-12 pm, and there is a bimodal size distribution. In the IAN of rats aged 30 months, D reaches up to some 12 pm, and there are two distinct peaks (Fig. 6). In comparison with that of younger adult rats, however, the size distribution has been shifted to the left. Separate measurements in adult animals (aged 3-9 months) show that myelinated fibres in the MN have a size range of 1-8 pm and a weakly bimodal pattern, with a major peak at 3 pm. Myelinated fibres in the IDN (including the M, branch) present a similar size range, and there are two peaks, one at 2 pm and one at 5 pm (Fig. 7). The proportion of large myelinated fibres is clearly higher in the IDN than in the MN. In the specific M, branch, D values vary from 1to 10 pm, and the size distribution is unimodal, with a peak a t 3 pm (Fig. 7). DISCUSSION
The rat dentition differs from the cat dentition in several important respects. In the diphyodont cat, a
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Fig. 2. Schematic drawing of the inferior dental nerve and its branches as well as their relation to the mandibular teeth seen from the lingual side. The mental nerve has been removed. The square indicates the part of the mandible detailed in the main figure. c, Branch to the third molar; d, branch to the odontogenic root zone of the incisor; e, branch to the second molar; f, branches to the midportion of the incisal periodontium; g, branches to the first molar; h, branch to the distal periodontium of the incisor. The thickness of the different branches have not been properly scaled in this scheme.
Fig. 3. a+: These representative drawings were made on the basis of light microscopic examination of semithin sections from the levels indicated by arrows labelled 3a, 3b, and 3c in Figure 2. The incisor is seen in the lower part of each picture. In b, a portion of the proximal root of the second molar is seen in the upper part. MN, mental nerve; IDN, inferior dental nerve.
primary dentition is established during the first postnatal weeks. Some months after birth, the primary dentition is shed and replaced by a permanent dentition, which reaches a fully grown stage and then gradually deteriorates (Fried, 1982). In the monophyodont
rat, on the other hand, the molars and the incisor erupt without predecessors. The rudimentary incisor that occurs in some rat strains belongs to the same tooth setup (Moss-Salentijn, 1975). In both species, the IAN is the major nerve projecting to the mandibular teeth. The
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Fig. 4. These electron micrographs show the appearance of transverse sections through the inferior alveolar nerve a t the level of the mandibular foramen in rats of different ages: a: fetal day 18, b 3 days after birth, c: 20 days after birth, d adult (9 months). x 5,100.
n
Fig. 5. Left. This diagram illustrates the total number of axons in the IAN from fetal day 17 to the age of 30 months. The dashed vertical line indicates birth. Each dot represents the number of axons in one nerve. Right: This graph shows the proportion of myelinated axons in
the rat IAN from fetal day 17 to 30 months after birth. Each dot represents the percentage of myelinated axons in the IAN of one rat. Both graphs refer to the level of the mandibular foramen.
developmental and adult properties of this nerve have been the topic of several careful studies in the cat (Fried and Hildebrand, 1981a,b, 1982a,b), but the course, composition, and development of the rat IAN are less well known. The present study represents the
most detailed morphological analysis of the rat IAN reported so far. Our gross anatomy results are in general agreement with previous descriptions (Rehak, 1963; Greene, 1968; Hebel and Stromberg, 1986). We confirm the observa-
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Fig. 6. These histograms show the fibre size distribution of myelinated axons in the rat IAN at the level of the mandibular foramen a t different ages as indicated [diameter (D), including myelin sheaths]. Note the different scales on the Y axis: 7-30 days vs. 70 days to 30 months.
tion that the IAN splits into an MN portion and an IDN descriptions of a single branch to each molar represent portion soon after entering the mandibular canal and simplifications (Rehak, 1963; Greene, 1968; Hebel and that the IDN sends a recurrent loop to the odontogenic Stromberg, 1986); actually, the molar branches are root zone of the incisor as well as some periodontal split into several fascicles when they leave the IDN. branches to its mid portion (Rehak, 1963). The early The early bifurcation of the rat IAN into MN and IDN emergence of a separate branch to the third molar and portions offers a possibility for separate experimental the presence of period.onta1branches to rostra1 portions studies on dental and mucosallcutaneous components. Cat tooth pulps contain trigeminal afferents as well of the incisor have not been reported before. Previous
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Fig. 7.Representative histograms illustrating the fibre size distribution (D, myelin sheaths included) of myelinated axons in the mental nerve (MN), the inferior dental nerve (IDN), and the branch to the third molar (M,) of the young adult rat.
as efferent sympathetic axons (Matthews and Robinson, 1980). The sympathetic innervation of cranial structures originates from the superior cervical ganglia and tends to follow blood vessels to the target areas (Matthews and Robinson, 1980). Thus tiny fascicles of unmyelinated and small myelinated axons can be observed along the inferior alveolar artery of the cat (Christensen, 1940). Rat teeth likewise have a sympathetic innervation (Larsson and Linde, 1971; Byers, 1984, 1985). Our observation that vessel-related bundles of C and A delta fibres are lacking in the rat inferior alveolar neurovascular bundle suggests that tooth-related sympathetic axons may travel within the IAN. However, the total axon number is not significantly reduced in nerves from rats subjected to chemical sympathectomy. This indicates that sympathetic axons destined to rat mandibular teeth are very few compared with the total number of IAN axons, that such axons are not affected by guanethidine treatment, or that they do not reach the teeth through the inferior alveolar neurovascular bundle. It has recently been reported that blood vessels in the lower lip and mandibular tooth pulps of the rat are innervated also by parasympathetic axons (Kaji et al., 1991). Whether these axons travel within the IAN is unknown. In agreement with previous observations (Holje et al., 19831, the adult rat IAN was found to contain -70% myelinated axons. Somewhat lower levels have been reported by Sugimoto and coworkers (1983). This proportion is higher than in the rat infraorbital nerve (Jacquin et al., 1984) and much higher than that found in, e.g., the rat sural nerve (Jenq and Coggeshall, 1985). A comparatively high percentage of myelinated axons has also been found in the cat IAN (Fried and Hildebrand, 1982a). The size range of adult rat IAN axons extends to 12 pm, and the diameter spectra exhibit a distinct bimodal pattern. Roughly similar data have been reported for the cat IAN (Fried and Hildebrand, 1982a). In the cat, the morphology of the mental nerve is similar to that of the entire IAN (Fried, 1984). In the rat, on the other hand, the proportion of myelinated axons is 65%in the MN and over 80%in the IDN. Obviously, tooth-related axons are myelinated to a greater extent than axons destined to skin and mucosa. Examination of the fibre sizes in the MN and the IDN shows similar size ranges but different size distributions. While small myelinated fibers predominate in the MN, the IDN also contains many large myelinated fibres. The average percentage of myelinated fibres with diameters 2 5 p,m is 22% in the MN and 37% in
the IDN. Surprisingly, our measurements reveal a majority of small myelinated fibres in the M3 branch. This pattern, which is consistent in all the nerves examined, may be explained by the fact that the M, branch has been examined at a level fairly close to the M, tooth (2-3 mm), whereas the IDN was examined at a greater distance from its target area. We conclude that the picture gained from examination of the IAN as a whole represents a combination of two different fibre populations. Our countings indicate that the developing rat IAN contains more axons prenatally and neonatally than at later postnatal stages. From 1 week after birth throughout postnatal life, the number of axons varies around 9,000. Similarly, the rat infraorbital nerve, which contains some 42,000 axons at birth, looses about 10,000 axons during early postnatal development (Renehan and Rhoades, 1984). Studies on other systems suggest that the elimination of superfluous axons during early development occurs in relation to establishment of target contact (Aguayo et al., 1973; Cowan, 1978). The mandibular dentition of the neonatal rat is highly incomplete, and no tooth has yet erupted (Mellanby, 1939; Bhaskar, 1953; Park, 1973). The incisor, the MI, and the M2 tooth germs are visible by X-ray, but the M, anlage has not reached beyond the dental lamina stage (Bhaskar, 1953; Johansson and Hildebrand, 1990). Whether the unerupted mandibular teeth are innervated is unknown. The most likely possibility is that pulpal and periodontal axons are lacking at this early stage. Accordingly, the IAN of the cat completes the elimination of superfluous axons long before the formation of its dental targets (Fried and Hildebrand, 1982a). It is well established that the rat incisor continues to grow throughout life, in order to compensate for the attrition caused by gnawing. Growth rates are reported to be some 0.5 m d d a y (Chiba et al., 1973). Tooth growth includes generation of new blood vessels (Pitaru et al., 1982) as well as growth and degeneration of pulpal axons (Bishop, 1981). We have not seen any features in the adult rat IAN trunk that may be linked to the de- and regenerative events a t terminal levels in the incisor. In the cat, the de- and regenerative neural changes associated with the developmental shift from the primary to the permanent dentition likewise do not involve the stem axons in the IAN (Fried and Hildebrand, 1981b). It seems likely that the neural remodelling associated with incisor growth is a preterminalterminal event, sparing the stem axons in the IAN.
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Also, the molars of the rat continue to grow in adult- koping. We thank Ms. Kristina Warnborg and Ms. hood. Addition of cement to the roots results in a slow Gunilla Oqvist for their excellent technical assistance. eruption that compensates for the successive wearing of the crown (Hoffman and Schour, 1941). It seems posLITERATURE CITED sible that this is accompanied by a gradual shrinkage Aguayo, A.J., L.C. Terry, and G.M. Bray 1973 Spontaneous loss of of the pulpal space, as secondary dentin is formed in axons in sympathetic unmyelinated nerve fibres of the rat during development. Brain Res., 54:360-364. the pulpal horns (Schour and Massler, 1949). Together J., and S. Gobel 1981 An HRP study of the central projecwith an increased occurrence of dental pathology dur- Arvidsson, tions of primary trigeminal neurons which innervate tooth pulps ing ageing (Miles, 1961) these processes could contribin the cat. Brain Res., 210:l-16. ute to a gradual age-related target deprivation of tooth- Arwill, T., L. Edwall, J . Lilja, L. Olgart, and S.-E. Svensson 1973 Ultrastructure of nerves in the dentinal-pulp border zone after related IAN parent neurons, as seems to be the case in sensory and autonomic nerve transection in the cat. Acta Odont. the cat and in man (see Fried, 1987). Nevertheless, the Scand., 3lt273-281. number of IAN axons remains unchanged late in life. Bhaskar, S.N. 1953 Growth pattern ofthe rat mandibule from 13 davs In spinal nerves, axonal atrophy, nodal widening, and insemination age to 30days after birth. Am. J. Anat., 92:l-55. various degrees of demyelination may occur as concom- Bishop, M.A. 1981 A fine-structural survey of the pulpal innervation in the rat mandibular incisor. Am. J . Anat., 160:213-229. itants to target deprivation (Gillespie and Stein, 1983; T., C. Hildebrand, and S. Berglund 1986 Nodes of Ranvier Brismar et al., 19861. Hence, it seems possible that the Brismar, above a neuroma in the rat sciatic nerve: Voltage clamp analysis shift of the fibre size distribution towards smaller sizes and electron microscopy. Brain Res., 378: 347-356. (present study) and the increasing occurrence of de- Byers, M.R. 1984 Dental sensory receptors. Int. Rev. Neurobiol., 25: 39-94. and remyelination in the old rat IAN (Hildebrand and M.R. 1985 Terminal arborization of individual sensory axons Johansson, 1991) to some extent may be secondary t o Byers, in dentin and pulp of rat molars. Brain Res., 345:181-185. regressive dental changes. Byers, M.R., and G.R. Holland 1977 Trigeminal nerve endings in gingiva, junctional epithelium and peridontal ligament of rat Myelination of the rat IAN begins a t birth (present molars as demonstrated by autoradiography. Anat. Rec., 188: study; Sugimoto et al., 1983). Already, 3-4 weeks 509-524. later, a close-to-adult level has been reached. This is Chad, D., W.G. Bradley, G. Rasool, P. Good, S. Reichlin, and J . Zivin much earlier than reported by Sugimoto and coworkers 1983 Sympathetic postganglionic unmyelinated axons in the rat peripheral nervous system. Neurology, 332341447. (1983). Examination of their Figure 3A shows, howM., M. Tsuruta, and K. Eto 1973 A photographic method of ever, that a nearly adult proportion of myelinated ax- Chiba, measuring eruption rates of rat mandibular incisors. Arch. Oral ons actually has been reached by 3-4 weeks. At this Biol., 18:1003-1010. age the incisor, MI, and M, reach functional occlusion, Christensen, K. 1940 Sympathetic nerve fibers in the alveolar nerves and nerves of the dental pulp. J . Dent. Res., 19:227-242. and the rat has begun to chew solid food (Mellanby, W.M. 1978 Aspects of neural development. Int. Rev. Physiol., 1939; Bhaskar, 1953; O’Brien et al., 1958; Park, 1973). Cowan, 17:149-191. Hence, by 3-4 weeks, the dental target area of the IAN Fried, K. 1982 Development, degeneration and regeneration of nerve is almost fully mature, except for M,, the development fibres in the feline inferior alveolar nerve and mandibular incisor pulps. Acta Physiol. Scand. Suppl. 504. of which is complete about 5 weeks postnatally (MelK. 1984 Structural development of the feline mental nerve. lanby, 1939; Park, 1973). Similarly, the end of de novo Fried, Anat. Rec., 210t347-355. myelination of the feline IAN coincides with comple- Fried, K. 1987 Changes in innervation of dentine and pulp with age. tion of the primary dentition and cessation of the weanFronteirs Oral Physiol., 6:63-84. Fried, K., and C. Hildebrand 1981a Developmental growth and deling period (Fried and Hildebrand, 1982b). generation of pulpal axons in feline primary incisors. J . Comp. The size distribution of myelinated rat IAN fibres Neurol., 203.37-51. shows a unimodal pattern during the period when the Fried, K., and C. Hildebrand 1981b Pulpal axons in developing, mateeth erupt (Mellanby, 1939; Park, 1973; Hebel and ture and aging feline permanent incisors. A study by electron microscopy. J. Comp. Neurol., 203t23-36. Stromberg, 1986). After 2 months, the pattern has become weakly bimodal. The larger myelinated axons Fried, K., and C. Hildebrand 1982a Axon number and size distribution in the developing feline inferior alveolar nerve. J . Neurol. continue to grow in size during young and middle-aged Sci., 53,169-180. adult life and tend to become smaller in the old adult Fried, K., and C. Hildebrand 19821, Qualitative structural developrat. Hence there appears not to be any simple relation ment of the feline inferior alveolar nerve. J. Anat., 134,517-531. between dental or behavioral maturation, on the one Fried, K., C. Hildebrand, and G. Erdelyi 1982 Myelin sheath thickness and internodal length of nerve fibres in the developing feline hand, and the development of the fibre size distribuinferior alveolar nerve. J . Neurol. Sci., 54:47-57. tion, on the other. A lack of direct coupling between Gillespie, M.J., and R.B. Stein 1983 The relationship between axon size growth and function has also been noted in the cat diameter, myelin thickness and conduction velocity during atrophy of mammalian peripheral nerves. Brain Res., 259:41-56. IAN (Fried and Hildebrand, 1982131. In conclusion, the present report describes the gross Greene, E.C. 1968 Anatomy of the Rat. Hafner Publishing Company, New York, Vol. 27. anatomy of the rat inferior alveolar neurovascular Hebel, R., and M.W. Stromberg 1986 Anatomy and Embryology of the bundle, analyses the content of unmyelinated and myLaboratory Rat. BioMed Verlag, Worthsee. elinated axons in the adult IAN and its major Hildebrand, C., and C.S. Johansson 1991 Nodal spacing in the developing, young adult and aging rat inferior alveolar nerve. Dev. branches, and presents data on the pre- and postnatal Brain Res., 64:175-181. maturation of this nerve. These data provide a basis for Hoffman, M.M., and I. Schour 1940 Quantitative studies in the defurther studies on developmental tooth-nerve interacvelopment of the rat molar. 11. Alveolar bone, cementum and tions. eruption (from birth to 500 days). Am. J. Orthodont., 26:854-874. ACKNOWLEDGMENTS
This study was supported by grants from the Swedish Medical Research Council (project 3761) and the research foundations of the University Hospital of Lin-
Holje, L., C. Hildebrand, and K. Fried 1983 Proportion of unmyelinated axons in the rat inferior alveolar nerve and mandibular molar pulps after neonatal administration of capsaicin. Brain Res., 266:133-136. Jacquin, M.F., A. Hess, G. Yang, P. Adamo, M.F. Math, A. Brown, and R.W. Rhoades 1984 Organization of the infraorbital nerve in rat:
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A quantitative electron microscopic study. Brain Res., 290:131135. Jenq, C.-B., and R.E. Coggeshalll985 Numbers of regenerating axons in parent and tributary peripheral nerves in the rat. Brain Res., 326:27-40. Johansson, C.S., and C. Hildebrand 1990 Development of nerves and teeth in the rat mandible., SOC.Neurosci., Abstracts, 16:829. Johnsson, E.M., Jr., and P.T. Manning 1984 Guanethidine-induced destruction of sympathetic neurons. Int. Rev. Neurobiol., 25:l37. Kaji, A,, T. Maeda, and S. Watanabe 1991 Parasympathetic innervation of cutaneous blood vessels examined by retrograde tracing in the rat lower lip. J . Autonom. Nerv. Syst., 32:153-158. Karlsson, U., D. Johnsen, and A.M. Herman 1974 Early degenerative nerve alterations in feline resorbing deciduous incisors as observed by electron microscopy. J . Dent. Res., 53t1428-1431. Karnes, J., R. Robb, P.C. O’Brien, E.H. Lambert, and P.J. Dyck 1977 Computerized image recognition for morphometry of nerve attribute of shape of sampled transverse sections of myelinated fibers which best estimates their average diameter. J. Neurol. Sci., 34:43 -51. Larsson, P.-A,, and A. Linde 1971 Adrenergic vessel innervation in the rat incisor pulp. Scand. J . Dent. Res., 79:7-12. Lumsden, A.G.S., and A.M. Davies 1983 Earlist sensory nerve fibres are guided to peripheral targets by attractants other than nerve growth factor. Nature, 306:786-788. Lumsden, A.G.S., and A.M. Davies 1986 Chemotropic effect of specific target epithelium in the developing mammalian nervous system. Nature, 323538-539. Matthews, B., and P.P. Robinson 1980 The course of post-ganglionic sympathetic fibres distributed with the trigeminal nerve in the cat. J . Physiol., 303:391-401. Mellanby, H. 1939 The development of teeth in the albino rat. Br. Dent. J., 66:76-86. Miles, A.E.W. 1961 Ageing in the teeth and oral tissues. In: Structural Aspects of Ageing. G.H. Bourne, ed. Pitman, London, pp. 352-397. Mohamed, S.S., and M.E. Atkinson 1983 A histological study of the innervation of developing mouse teeth. J . Anat., 136:735749.
Moss-Salentijn, L. 1975 Studies on dentin. 2. Vestigial lacteal incisor teeth of the rat. Acta anat., 92:329-350. Naftel, J.P. 1987 Sympathetic neuronotrophic activity in the pulp of the cat canine tooth. Arch. Oral Biol., 32:897-905. OBrien, C., S.N. Bhaskar, and A.G. Brodie 1958 Eruptive mechanism and movement in the first molar of the rat. J . Dent. Res., 37: 467-484. Olgart, L., T. Hokfelt, G. Nilsson, and B. Pernow 1977 Localization of substance P-like immunoreactivity in nerves in the tooth pulp. Pain, 4:153-159. Park, A.W. 1973 The maxillary and mandibular osteodental fissures of the laboratory rat (Rattus norvegicus) in relation to molar eruption. Anat. Anz. Bd., 133:125-137. Pearson, A.A. 1977 The early innervation of the developing deciduous teeth. J. Anat., 123.563-577. Pitaru, S., Y. Michaeli, and G. Zajicek 1982 Vascular turnover of a continuously growing organ: The rat incisor. Anat. Rec., 202: 203-208. Rehak, J.R. 1963 Course and resection of the inferior alveolar nerve in the albino rat. J. Dent. Res., 42:1159-1168. Renehan, W.E., and R.W. Rhoades 1984 A quantitative electron microscopic analysis of the rat infraorbital nerve in the newborn rat. Brain Res., 322:369-373. Ridehalgh, E., and D. Stewart 1938 The course of the incisor branch of the inferior dental nerve in rodents and some observations on the nerve supply of the pulp. J. Anat., 72416-421. Robinson, P.P. 1979 The course, relations and distribution of the inferior alveolar nerve and its branches in the cat. Anat. Rec., 195: 265-272. Robinson, P.P. 1981 Reinnervation of teeth, mucous membrane and skin following section of the inferior alveolar nerve in the cat. Brain Res., 220:241-253. Schour, I., and M. Massler 1949 The teeth. In: The Rat in Laboratory Investigation. E.J. Farris and J.Q. Griffth Jr., eds. J.B. Lippincott Company, Philadelphia, pp. 104-165. Sugimoto, T., M. Sugita, M. Takemura, S. Kitamura, and A. Sakai 1983 The postnatal development of the rat inferior alveolar nerve. J . Osaka Univ. Dent. School, 2359-71. Warshawsky, H., and G. Moore 1967 A technique for the fixation and decalcification of rat incisors for electron microscopy. J. Histochem. Cytochem., 1 5 5 4 2 4 4 9 .
Anatomy and Developmental Chronology of the Rat Inferior Alveolar Nerve CARINA S. JOHANSSON, CLAES HILDEBRAND, AND BO POVLSEN Departments of Cell Biology (C.S.J.,C.H.) and Hand and Plastic Surgery (B.P.), Faculty of Health Sciences, University of Linkoping, 581 85 Linkoping, Sweden
ABSTRACT
This report describes the anatomy of the inferior alveolar neurovascular bundle in the adult rat and provides a quantitative analysis of the developing inferior alveolar nerve (IAN). Soon after its entrance in the mandibular canal, the IAN splits into a mental nerve (MN) and a n inferior dental nerve (IDN), which course in separate bony compartments. The MN passes unbranched through the mandibular canal. The IDN sends branches to the incisor, the first molar, and the second molar. The third molar (M,) is supplied by a separate IAN branch. The adult rat IAN contains 8,000-10,000 axons, 70%of which are myelinated. The MN accounts for 70%of all IAN axons, the IDN 26%,and 4%form the M, branch. The proportion of large myelinated axons is lower in the MN than in the IDN. Following chemical sympathectomy, the IAN axon number does not change in a statistically significant way. The total number of IAN axons, which is high prenatally and neonatally, has decreased to the adult level about 1 week after birth. De novo myelination commences at birth and is complete 3-4 weeks later. The size spectrum of the myelinated fibres is narrow and unimodal during the first postnatal weeks. By 1 month, the largest fibres reach diameters of -6 pm, and a bimodal pattern is emerging. From 3 months and on, the size range reaches up to 10-12 pm, and the distribution is bimodal. These data provide a basis for further studies on developmental tooth-nerve interactions. o 1992 Wiley-Liss, Inc.
Vertebrate teeth have a rich sensory innervation, and the dental pulp is a well-defined target structure, supplied by a fairly homogeneous nerve fibre population. From a morphogenetic point of view, the trigeminal branches projecting to the teeth are of particular interest, because of the ontogenetically late establishment of dental target contacts, the extensive developmental remodelling of the dentition in diphyodont and polyphyodont species, and the progressive tooth alterations during ageing (Miles, 1961; Fried and Hildebrand, 1981a,b; Mohamed and Atkinson, 1983, Fried, 1987). Much experimental work on tooth-nerve relations has been performed in the cat (Arwill e t al., 1973; Karlsson et al., 1974; Olgart et al., 1977; Robinson, 1979, 1981; Arvidsson and Gobel, 1981). Some studies describe tooth-nerve relations in the rat or the mouse (Byers and Holland, 1977;Pearson, 1977; Bishop, 1981; Mohamed and Atkinson, 1983; see Byers, 1984, 1985). Few reports deal with the basic biological mechanisms involved in the establishment and maintenance of tooth nerves. For such studies, the use of developing cellular explants in vitro might represent a rewarding approach (cf Lumsden and Davies, 1983, 1986; Naftel, 1987). From various points of view, the rat is more suitable than the cat for in vitro experiments on explanted tissues. Since the mandibular dentition is supplied by a single nerve trunk-the inferior alveolar nerve (IAN) (Greene, 1968; Hebel and Stromberg, 1986; Robinson, 1979)-it appears suitable for experimental studies. The anatomy and development of the feline IAN is elucidated in several studies (Robinson, c
1992 WILEY-LISS. INC
1979; Fried and Hildebrand, 1982a,b; Fried et al., 1982), but the rat IAN is less well known. The few anatomical descriptions available are rather incomplete (Ridehalgh and Stewart, 1938; Rehak, 1963). Apart from a recent report from our laboratory (Hildebrand and Johansson, 1991), we are aware of only one paper that deals with the developmental maturation of this nerve (Sugimoto et al., 1983). Morphological and maturational data on the rat IAN are needed as a basis for studies on developmental tooth-nerve interactions. Hence, the aim of the present work is to describe the anatomy of the inferior alveolar neurovascular bundle in the adult rat and to provide a chronology for the structural development of the rat IAN. Some of these results have been reported in abstract form (Johansson and Hildebrand, 1990). MATERIALS AND METHODS Dissection
Adult male and female Wistar and Sprague-Dawley rats were anaesthetized with ether and exsanguinated (n = 20). The mandible was removed, split in halves, and cleaned from soft tissues so that the mandibular and mental foramina and their contents became visible. The inferior alveolar neurovascular bundle was exposed by opening the mandibular canal from the buccal side, using a dental bur and fine rongeurs. The
Received September 12, 1991; accepted December 14, 1991.
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contents of the bundle were subjected to some blunt dissection and were examined in a Nikon SMZ-2T dissection microscope equipped with a Nikon F-301 camera. Immersion of the dissected specimen in saline simplified identification of small branches. Chemical Sympathectomy
Rat pups, of both sexes, were injected under the dorsal skin with a 1.5% solution of guanethidine sulphate (Sigma) in sterile water (50 mg/kg, pH adjusted to 7.4), 6 days per week for 3 weeks, beginning 3 days after birth (n = 4). This treatment destroys most neurons in the sympathetic ganglia (Chad et al., 1983, Johnson and Manning, 1984). These animals were allowed to survive for 3-6 months. At that age, they were perfused and the IAN was collected and prepared for electron microscopic examination as described below. Nerves from untreated age-matched animals used for the developmental part of this study (vide infra) served as controls (n = 6). Light Microscopy (LM)
To reach a satisfactory description of the various thin branches included in the inferior alveolar neurovascular bundle, it was necessary to perform an LM analysis of serial sections. For this purpose, adult rats were anaesthetized with chloral hydrate (30 mg/100 g i.p.) and perfused with glutaraldehyde, as described below (n = 5). After perfusion, the mandible was removed, postfixed, and rinsed in cold buffer. It was split in halves and cleaned from soft tissues. The crowns of the molars and part of the alveolar process were removed. Specimens composed of the portion of the mandibular corpus that contains the mandibular canal were demineralized in 4% EDTA (Sigma) in cacodylate buffer (pH 7.2-7.4,4" C, 6-8 weeks) (Warshawsky and Moore, 1967). The EDTA solution was changed three times weekly. The demineralized specimens were dehydrated in alcohol and embedded in JB4 resin (Bio-Rad).Transverse serial sections (3 Fm) were cut from the mandibular canal region with an LKB Historange microtome, using glass knives. Sections with a spacing of 0.1 mm were mounted in sequential order on glass slides, stained with toluidine blue, and examined in a light microscope. Drawings showing the different branches a t selected levels were made with the aid of a drawing tube. Electron Microscopy (EM)
Material intended for EM was collected from 13 rat fetuses aged fetal day 17-21 and from 38 postnatal rats aged from newborn to 30 months. Fetuses and young rat pups (0-5 days) were anesthetized by hypothermia. Older pups and adult rats were anesthetized with chloral hydrate. The rats were perfused with Tyrode's solution, followed by 5% glutaraldehyde dissolved in a 300 mOsm phosphate buffer, also containing 0.1 M sucrose. Specimens were taken from the IAN at its entrance in the mandibular canal, excluding the mylohyoid branch. The specimens were postfixed in 5% phosphate-buffered glutaraldehyde, rinsed in the buffer, osmicated in 2% buffered OsO,, and rinsed. After dehydration in acetone, the specimens were embedded in Vestopal W. Semithin toluidine blue-stained cross sections from the IAN were examined in the light
microscope for orientation. Thin cross sections were cut on an LKB Ultrotome I11 or IV, using glass or diamond knives. The sections were collected on one-hole copper grids coated with formvar. The thin sections were photographed in a Jeol 1200EX electron microscope. When dealing with the completely unmyelinated fetal nerves, all axons present in approximately one-half the cross-cut nerve were counted. After dividing the total area of the cross-cut nerve into squares, with the size of one microscopic field, every second square was photographed at x 15,000. The axons on the micrographs were counted, and the areas they occupied were measured with a Videoplan equipment (Kontron; Zeiss). This rocedure resulted in an average number of axons/Fm . By measuring the total area of the cross-cut nerve, the total number of axons could be estimated. With respect to nerves from rat pups younger than 4 days old, a t which time few myelinated axons have formed, every fourth square was processed as described above, and the total number of axons was determined in the same way. In nerves from older animals, the number of myelinated axons was obtained from montages of electron micrographs covering the entire nerve ( x 4,000-6,000). For determination of unmyelinated axon numbers, the sections were reexamined in the EM and all unmyelinated axons surrounding each of some 1,000 myelinated axons were counted. In each nerve, a t least five different areas, together covering approximately one-eighth of the total nerve area, were used for counting. The cross-sectional areas of - 1,000 myelinated fibres (myelin sheaths included) were measured on EM prints ( x 2,000-4,000) showing IAN cross sections from eight different postnatal ages (one animal/age), using a Videoplan equipment (Kontron; Zeiss). Fibre diameters were calculated from area values, assuming that they correspond to circle diameters (Karnes et al., 1977). In addition to countings and measurements on the adult IAN as a whole, the dental and mental branches, and the specific branch to the third molar were considered separately in three cases. These data were obtained from within 1mm distal to the bifurcation of the IAN into the mental nerve and the inferior dental nerve.
f
RESULTS Contents of the Mandibular Canal
The IAN courses along the lingual aspect of the mandibular ramus and emits the mylohyoid branch some 8 mm before the mandibular foramen, which it enters as a single coherent branch (Fig. 1).One artery and some small veins accompany the nerve, together forming the inferior alveolar neurovascular bundle (Fig. 3a). We could not see any separate bundles of small myelinated and unmyelinated axons in association with the blood vessels. Shortly after its entrance in the mandibular canal, the IAN emits a slender branch, which splits into three fascicles for the roots of the third molar (M3) (Figs. 1, 2, 3a). Below M,, the IAN splits into its two major branches, the mental nerve (MN) and the inferior dental nerve (IDN), the former being thicker than the latter (Fig. 1). Shortly after this nerve division, the mandibular canal bifurcates into a buccal portion, containing the MN, and a lingual portion, containing the
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well as in the young and old adult stages, axon number varies between 8,000 and 10,000 (Fig. 5). Separate counts in the MN and the IDN of adult rats showed that the average MN contains 6,250 axons (70%of all IAN axons), while the average IDN contains 2,630 axons (30%). The latter figure includes the branch to M,, which consists of 348 axons (4%). In the rats subjected to chemical sympathectomy, the average total number of axons was found to be 7,400 (k890; SD). The six age-matched control nerves contain 8,800 axons (5460; SD). Statistical analysis through the Wilcoxon rank-sum test showed that there is no significant difference between the guanethidine treated rats and the control group. Proportion of Myelinated Axons
Fig. 1. Photograph showing a dissected rat inferior alveolar nerve (IAN) and some of its branches: a, IAN nerve trunk; b, mental nerve; c, branch to the third molar; d, branch to the odontogenic root zone of the incisor; e, branch to the second molar; f, branch to the midportion of the incisal periodontium; g, branches to the first molar. X 30.
IDN (Fig. 3b). The bony septum that separates the two portions begins at a level between the M, and the second molar (M,). The MN extends unbranched through the buccal portion of the canal, exits through the mental foramen, and sends branches to the mucosa and skin of the lower lip and to the skin of the chin. The cutaneous branches cover an area of about 25 mm'. The IDN courses rostrally towards and into the lingual compartment of the mandibular canal, emitting a series of branches (Fig. 2). Shortly after its appearance as a separate nerve trunk, a thin recurrent loop leaves the IDN and courses to the odontogenic root zone of the incisor (Figs. 1, 2,3a). This twig, which is composed of a majority of unmyelinated axons and some four or five myelinated ones, is so thin that it can barely be discerned a t dissection. Below the M,, a branch with two fascicles courses rostrally towards the periodontium and the root apices of M, (Figs. 2, 3b). In the average rat, a tiny ventral branch composed of a few fascicles leaves the IDN below M, and enters the periodontium of the incisor (Fig. 2). In the individual case, the origin of this branch varies from below M, to below the first molar (MI). The small nerve fascicles to the periodontium of the incisor are composed of comparatively large myelinated axons (diameter = 8-9 pm) and a few unmyelinated axon bundles. The terminal branches of the IDN include two large and several small ramifications to the MI. A minor twig from the IDN courses to the most rostra1 periodontium of the incisor (Figs. 2, 3c). Number of Axons
Representative examples of the EM appearance of the IAN at some developmental stages are shown in Figure 4. In the youngest fetal rats examined (fetal day 17), the average IAN contains about 13,700 axons (range 11,400-16,500; Fig. 5). Subsequently, the number decreases, reaching a mean level of -11,100 at birth. The decrease has ceased 1 week postnatally, when the average number is about 8,800 (range 8,0009,400; Fig. 5). During the continued development, as
Throughout prenatal development, the IAN is composed of unmyelinated axons (Fig. 5).Myelination commences on the day of birth. Subsequently, the proportion of myelinated axons increases very rapidly, particularly during the second week (Fig. 5). One week postnatally this proportion is -15%, and by 10 days about one half of all axons have achieved myelin sheaths. From 3-4 weeks on, the proportion of myelinated axons varies around 70% (Fig. 5). Separate counts show that the proportion of myelinated axons is 65% in the MN and 81% in the IDN (including the M, branch). The separate branch to M, contains 67% myelinated axons. In chemically sympathectomized animals, the proportion of myelinated IAN axons is 69%, i.e., similar to that in normal nerves. Size Distribution of Myelinated Fibres
One week after birth, the myelinated IAN fibres exhibit a unimodal size spectrum with a diameter (D) range of 0.8-3 pm (Fig. 6). During the second week, the upper end of the size spectrum shifts to -4 pm. After 3 weeks, the maximum diameter is -6 pm, and there is a single peak a t 3 pm (Fig. 6). By 1 month, the size range extends from 1 to 6 pm, and the pattern is weakly bimodal. At about 2 months, D ranges from 1to 9 pm, and the pattern is bimodal (Fig. 6). In adult rats aged 3-12 months, the diameter range extends from 1 to 10-12 pm, and there is a bimodal size distribution. In the IAN of rats aged 30 months, D reaches up to some 12 pm, and there are two distinct peaks (Fig. 6). In comparison with that of younger adult rats, however, the size distribution has been shifted to the left. Separate measurements in adult animals (aged 3-9 months) show that myelinated fibres in the MN have a size range of 1-8 pm and a weakly bimodal pattern, with a major peak at 3 pm. Myelinated fibres in the IDN (including the M, branch) present a similar size range, and there are two peaks, one at 2 pm and one at 5 pm (Fig. 7). The proportion of large myelinated fibres is clearly higher in the IDN than in the MN. In the specific M, branch, D values vary from 1to 10 pm, and the size distribution is unimodal, with a peak a t 3 pm (Fig. 7). DISCUSSION
The rat dentition differs from the cat dentition in several important respects. In the diphyodont cat, a
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Fig. 2. Schematic drawing of the inferior dental nerve and its branches as well as their relation to the mandibular teeth seen from the lingual side. The mental nerve has been removed. The square indicates the part of the mandible detailed in the main figure. c, Branch to the third molar; d, branch to the odontogenic root zone of the incisor; e, branch to the second molar; f, branches to the midportion of the incisal periodontium; g, branches to the first molar; h, branch to the distal periodontium of the incisor. The thickness of the different branches have not been properly scaled in this scheme.
Fig. 3. a+: These representative drawings were made on the basis of light microscopic examination of semithin sections from the levels indicated by arrows labelled 3a, 3b, and 3c in Figure 2. The incisor is seen in the lower part of each picture. In b, a portion of the proximal root of the second molar is seen in the upper part. MN, mental nerve; IDN, inferior dental nerve.
primary dentition is established during the first postnatal weeks. Some months after birth, the primary dentition is shed and replaced by a permanent dentition, which reaches a fully grown stage and then gradually deteriorates (Fried, 1982). In the monophyodont
rat, on the other hand, the molars and the incisor erupt without predecessors. The rudimentary incisor that occurs in some rat strains belongs to the same tooth setup (Moss-Salentijn, 1975). In both species, the IAN is the major nerve projecting to the mandibular teeth. The
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Fig. 4. These electron micrographs show the appearance of transverse sections through the inferior alveolar nerve a t the level of the mandibular foramen in rats of different ages: a: fetal day 18, b 3 days after birth, c: 20 days after birth, d adult (9 months). x 5,100.
n
Fig. 5. Left. This diagram illustrates the total number of axons in the IAN from fetal day 17 to the age of 30 months. The dashed vertical line indicates birth. Each dot represents the number of axons in one nerve. Right: This graph shows the proportion of myelinated axons in
the rat IAN from fetal day 17 to 30 months after birth. Each dot represents the percentage of myelinated axons in the IAN of one rat. Both graphs refer to the level of the mandibular foramen.
developmental and adult properties of this nerve have been the topic of several careful studies in the cat (Fried and Hildebrand, 1981a,b, 1982a,b), but the course, composition, and development of the rat IAN are less well known. The present study represents the
most detailed morphological analysis of the rat IAN reported so far. Our gross anatomy results are in general agreement with previous descriptions (Rehak, 1963; Greene, 1968; Hebel and Stromberg, 1986). We confirm the observa-
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tion that the IAN splits into an MN portion and an IDN descriptions of a single branch to each molar represent portion soon after entering the mandibular canal and simplifications (Rehak, 1963; Greene, 1968; Hebel and that the IDN sends a recurrent loop to the odontogenic Stromberg, 1986); actually, the molar branches are root zone of the incisor as well as some periodontal split into several fascicles when they leave the IDN. branches to its mid portion (Rehak, 1963). The early The early bifurcation of the rat IAN into MN and IDN emergence of a separate branch to the third molar and portions offers a possibility for separate experimental the presence of period.onta1branches to rostra1 portions studies on dental and mucosallcutaneous components. Cat tooth pulps contain trigeminal afferents as well of the incisor have not been reported before. Previous
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Fig. 7.Representative histograms illustrating the fibre size distribution (D, myelin sheaths included) of myelinated axons in the mental nerve (MN), the inferior dental nerve (IDN), and the branch to the third molar (M,) of the young adult rat.
as efferent sympathetic axons (Matthews and Robinson, 1980). The sympathetic innervation of cranial structures originates from the superior cervical ganglia and tends to follow blood vessels to the target areas (Matthews and Robinson, 1980). Thus tiny fascicles of unmyelinated and small myelinated axons can be observed along the inferior alveolar artery of the cat (Christensen, 1940). Rat teeth likewise have a sympathetic innervation (Larsson and Linde, 1971; Byers, 1984, 1985). Our observation that vessel-related bundles of C and A delta fibres are lacking in the rat inferior alveolar neurovascular bundle suggests that tooth-related sympathetic axons may travel within the IAN. However, the total axon number is not significantly reduced in nerves from rats subjected to chemical sympathectomy. This indicates that sympathetic axons destined to rat mandibular teeth are very few compared with the total number of IAN axons, that such axons are not affected by guanethidine treatment, or that they do not reach the teeth through the inferior alveolar neurovascular bundle. It has recently been reported that blood vessels in the lower lip and mandibular tooth pulps of the rat are innervated also by parasympathetic axons (Kaji et al., 1991). Whether these axons travel within the IAN is unknown. In agreement with previous observations (Holje et al., 19831, the adult rat IAN was found to contain -70% myelinated axons. Somewhat lower levels have been reported by Sugimoto and coworkers (1983). This proportion is higher than in the rat infraorbital nerve (Jacquin et al., 1984) and much higher than that found in, e.g., the rat sural nerve (Jenq and Coggeshall, 1985). A comparatively high percentage of myelinated axons has also been found in the cat IAN (Fried and Hildebrand, 1982a). The size range of adult rat IAN axons extends to 12 pm, and the diameter spectra exhibit a distinct bimodal pattern. Roughly similar data have been reported for the cat IAN (Fried and Hildebrand, 1982a). In the cat, the morphology of the mental nerve is similar to that of the entire IAN (Fried, 1984). In the rat, on the other hand, the proportion of myelinated axons is 65%in the MN and over 80%in the IDN. Obviously, tooth-related axons are myelinated to a greater extent than axons destined to skin and mucosa. Examination of the fibre sizes in the MN and the IDN shows similar size ranges but different size distributions. While small myelinated fibers predominate in the MN, the IDN also contains many large myelinated fibres. The average percentage of myelinated fibres with diameters 2 5 p,m is 22% in the MN and 37% in
the IDN. Surprisingly, our measurements reveal a majority of small myelinated fibres in the M3 branch. This pattern, which is consistent in all the nerves examined, may be explained by the fact that the M, branch has been examined at a level fairly close to the M, tooth (2-3 mm), whereas the IDN was examined at a greater distance from its target area. We conclude that the picture gained from examination of the IAN as a whole represents a combination of two different fibre populations. Our countings indicate that the developing rat IAN contains more axons prenatally and neonatally than at later postnatal stages. From 1 week after birth throughout postnatal life, the number of axons varies around 9,000. Similarly, the rat infraorbital nerve, which contains some 42,000 axons at birth, looses about 10,000 axons during early postnatal development (Renehan and Rhoades, 1984). Studies on other systems suggest that the elimination of superfluous axons during early development occurs in relation to establishment of target contact (Aguayo et al., 1973; Cowan, 1978). The mandibular dentition of the neonatal rat is highly incomplete, and no tooth has yet erupted (Mellanby, 1939; Bhaskar, 1953; Park, 1973). The incisor, the MI, and the M2 tooth germs are visible by X-ray, but the M, anlage has not reached beyond the dental lamina stage (Bhaskar, 1953; Johansson and Hildebrand, 1990). Whether the unerupted mandibular teeth are innervated is unknown. The most likely possibility is that pulpal and periodontal axons are lacking at this early stage. Accordingly, the IAN of the cat completes the elimination of superfluous axons long before the formation of its dental targets (Fried and Hildebrand, 1982a). It is well established that the rat incisor continues to grow throughout life, in order to compensate for the attrition caused by gnawing. Growth rates are reported to be some 0.5 m d d a y (Chiba et al., 1973). Tooth growth includes generation of new blood vessels (Pitaru et al., 1982) as well as growth and degeneration of pulpal axons (Bishop, 1981). We have not seen any features in the adult rat IAN trunk that may be linked to the de- and regenerative events a t terminal levels in the incisor. In the cat, the de- and regenerative neural changes associated with the developmental shift from the primary to the permanent dentition likewise do not involve the stem axons in the IAN (Fried and Hildebrand, 1981b). It seems likely that the neural remodelling associated with incisor growth is a preterminalterminal event, sparing the stem axons in the IAN.
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Also, the molars of the rat continue to grow in adult- koping. We thank Ms. Kristina Warnborg and Ms. hood. Addition of cement to the roots results in a slow Gunilla Oqvist for their excellent technical assistance. eruption that compensates for the successive wearing of the crown (Hoffman and Schour, 1941). It seems posLITERATURE CITED sible that this is accompanied by a gradual shrinkage Aguayo, A.J., L.C. Terry, and G.M. Bray 1973 Spontaneous loss of of the pulpal space, as secondary dentin is formed in axons in sympathetic unmyelinated nerve fibres of the rat during development. Brain Res., 54:360-364. the pulpal horns (Schour and Massler, 1949). Together J., and S. Gobel 1981 An HRP study of the central projecwith an increased occurrence of dental pathology dur- Arvidsson, tions of primary trigeminal neurons which innervate tooth pulps ing ageing (Miles, 1961) these processes could contribin the cat. Brain Res., 210:l-16. ute to a gradual age-related target deprivation of tooth- Arwill, T., L. Edwall, J . Lilja, L. Olgart, and S.-E. Svensson 1973 Ultrastructure of nerves in the dentinal-pulp border zone after related IAN parent neurons, as seems to be the case in sensory and autonomic nerve transection in the cat. Acta Odont. the cat and in man (see Fried, 1987). Nevertheless, the Scand., 3lt273-281. number of IAN axons remains unchanged late in life. Bhaskar, S.N. 1953 Growth pattern ofthe rat mandibule from 13 davs In spinal nerves, axonal atrophy, nodal widening, and insemination age to 30days after birth. Am. J. Anat., 92:l-55. various degrees of demyelination may occur as concom- Bishop, M.A. 1981 A fine-structural survey of the pulpal innervation in the rat mandibular incisor. Am. J . Anat., 160:213-229. itants to target deprivation (Gillespie and Stein, 1983; T., C. Hildebrand, and S. Berglund 1986 Nodes of Ranvier Brismar et al., 19861. Hence, it seems possible that the Brismar, above a neuroma in the rat sciatic nerve: Voltage clamp analysis shift of the fibre size distribution towards smaller sizes and electron microscopy. Brain Res., 378: 347-356. (present study) and the increasing occurrence of de- Byers, M.R. 1984 Dental sensory receptors. Int. Rev. Neurobiol., 25: 39-94. and remyelination in the old rat IAN (Hildebrand and M.R. 1985 Terminal arborization of individual sensory axons Johansson, 1991) to some extent may be secondary t o Byers, in dentin and pulp of rat molars. Brain Res., 345:181-185. regressive dental changes. Byers, M.R., and G.R. Holland 1977 Trigeminal nerve endings in gingiva, junctional epithelium and peridontal ligament of rat Myelination of the rat IAN begins a t birth (present molars as demonstrated by autoradiography. Anat. Rec., 188: study; Sugimoto et al., 1983). Already, 3-4 weeks 509-524. later, a close-to-adult level has been reached. This is Chad, D., W.G. Bradley, G. Rasool, P. Good, S. Reichlin, and J . Zivin much earlier than reported by Sugimoto and coworkers 1983 Sympathetic postganglionic unmyelinated axons in the rat peripheral nervous system. Neurology, 332341447. (1983). Examination of their Figure 3A shows, howM., M. Tsuruta, and K. Eto 1973 A photographic method of ever, that a nearly adult proportion of myelinated ax- Chiba, measuring eruption rates of rat mandibular incisors. Arch. Oral ons actually has been reached by 3-4 weeks. At this Biol., 18:1003-1010. age the incisor, MI, and M, reach functional occlusion, Christensen, K. 1940 Sympathetic nerve fibers in the alveolar nerves and nerves of the dental pulp. J . Dent. Res., 19:227-242. and the rat has begun to chew solid food (Mellanby, W.M. 1978 Aspects of neural development. Int. Rev. Physiol., 1939; Bhaskar, 1953; O’Brien et al., 1958; Park, 1973). Cowan, 17:149-191. Hence, by 3-4 weeks, the dental target area of the IAN Fried, K. 1982 Development, degeneration and regeneration of nerve is almost fully mature, except for M,, the development fibres in the feline inferior alveolar nerve and mandibular incisor pulps. Acta Physiol. Scand. Suppl. 504. of which is complete about 5 weeks postnatally (MelK. 1984 Structural development of the feline mental nerve. lanby, 1939; Park, 1973). Similarly, the end of de novo Fried, Anat. Rec., 210t347-355. myelination of the feline IAN coincides with comple- Fried, K. 1987 Changes in innervation of dentine and pulp with age. tion of the primary dentition and cessation of the weanFronteirs Oral Physiol., 6:63-84. Fried, K., and C. Hildebrand 1981a Developmental growth and deling period (Fried and Hildebrand, 1982b). generation of pulpal axons in feline primary incisors. J . Comp. The size distribution of myelinated rat IAN fibres Neurol., 203.37-51. shows a unimodal pattern during the period when the Fried, K., and C. Hildebrand 1981b Pulpal axons in developing, mateeth erupt (Mellanby, 1939; Park, 1973; Hebel and ture and aging feline permanent incisors. A study by electron microscopy. J. Comp. Neurol., 203t23-36. Stromberg, 1986). After 2 months, the pattern has become weakly bimodal. The larger myelinated axons Fried, K., and C. Hildebrand 1982a Axon number and size distribution in the developing feline inferior alveolar nerve. J . Neurol. continue to grow in size during young and middle-aged Sci., 53,169-180. adult life and tend to become smaller in the old adult Fried, K., and C. Hildebrand 19821, Qualitative structural developrat. Hence there appears not to be any simple relation ment of the feline inferior alveolar nerve. J. Anat., 134,517-531. between dental or behavioral maturation, on the one Fried, K., C. Hildebrand, and G. Erdelyi 1982 Myelin sheath thickness and internodal length of nerve fibres in the developing feline hand, and the development of the fibre size distribuinferior alveolar nerve. J . Neurol. Sci., 54:47-57. tion, on the other. A lack of direct coupling between Gillespie, M.J., and R.B. Stein 1983 The relationship between axon size growth and function has also been noted in the cat diameter, myelin thickness and conduction velocity during atrophy of mammalian peripheral nerves. Brain Res., 259:41-56. IAN (Fried and Hildebrand, 1982131. In conclusion, the present report describes the gross Greene, E.C. 1968 Anatomy of the Rat. Hafner Publishing Company, New York, Vol. 27. anatomy of the rat inferior alveolar neurovascular Hebel, R., and M.W. Stromberg 1986 Anatomy and Embryology of the bundle, analyses the content of unmyelinated and myLaboratory Rat. BioMed Verlag, Worthsee. elinated axons in the adult IAN and its major Hildebrand, C., and C.S. Johansson 1991 Nodal spacing in the developing, young adult and aging rat inferior alveolar nerve. Dev. branches, and presents data on the pre- and postnatal Brain Res., 64:175-181. maturation of this nerve. These data provide a basis for Hoffman, M.M., and I. Schour 1940 Quantitative studies in the defurther studies on developmental tooth-nerve interacvelopment of the rat molar. 11. Alveolar bone, cementum and tions. eruption (from birth to 500 days). Am. J. Orthodont., 26:854-874. ACKNOWLEDGMENTS
This study was supported by grants from the Swedish Medical Research Council (project 3761) and the research foundations of the University Hospital of Lin-
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