Periapical innervation of the ferret canine and the local retrograde neural changes after pulpectomy.
код для вставкиСкачатьTHE ANATOMICAL RECORD 220:318-327 (1988) Periapical Innervation of the Ferret Canine and the Local Retrograde Neural Changes After Pulpectomy G.R. HOLLAND Division of Endodontics, Faculty of Dentistry, University ofAlberta, Edmonton, Alberta, Canada T6G 2N8 ABSTRACT The amputation of the dental pulp severs a population of axons that are predominantly in the A6 and C fiber size range and are principally involved in nociception. Local periapical neuromas, if they are formed after pulpectomy, may be the sites of spontaneous nervous activity that may, in some circumstances, be involved in the genesis of chronic pain. The periapical tissues from the mandibular canines of four ferrets were examined 3 months after pulpectomies. Silver-stained paraffin sections were examined in three dimensions a t the light microscope level. Ultrathin sections were examined a t the electron microscope level. Compared with contralateral and independent controls, the principal changes were the loss of the periodontal plexus around the root apex, the extension of damage well below the apical foramen, and the persistance of inflammation 12 weeks postoperatively. While a somewhat disorderly mass of nerve fibers develops subapically, the arrangement has only some of the features usually associated with neuromas. The tooth pulp has a rather unusual innervation. It contains dense populations of A6 and C fibers (Johnsen and Karlsson, 1974; Beasley and Holland, 1978), and the sensation experienced from various stimuli applied to it is pain (Anderson et al., 1970). While recent studies have shown that the pulp does in fact contain A6 fibers (Cadden et al., 1982; Holland and Robinson, 1983) and may be the source, under some circumstances, of sensations other than pain (Grusser et al., 1982), it is probably true that the predominant role of its innervation is nociceptive. Thus, when the dental pulp is destroyed, a preponderance of pain fibers are damaged. Tooth extraction or pulpectomy is effectively amputation of a specialized nociceptive population-a modality-specific form of deafferentation. The axons are severed 1-2 cm from their termination and their target organ is removed. It is possible that local changes after peripheral nerve damage may be involved in the genesis of chronic pain. Spontaneous activity originates in neuromas formed at the site of damage or from the sensory ganglion cells (Wall and Gutnik, 1974; Burchiel and Russell, 1985; Wall and Devor, 1983). Little is known about the local changes after damage to the nerves of the dental pulp. Hansen (1980) found the existence of small traumatic neuromas at the base of extraction sockets. Ratner et al. (1979) described the existence of small intrabony defects at the site of previous extractions in patients with trigeminal neuralgia. Histological examination of the contents of these defects shows neuroma-like tissue. After curettage of these areas the symptoms of trigeminal neuralgia are lost. No one has thus far looked at the possibility of neuroma formation after damage - limited to the dental pulp. The study reported here is a n initial step in examining the local neural changes after pulpectomy. 0 1988 ALAN R. LISS, INC. MATERIALS AND METHODS Five young adult ferrets 4-6 months old a t initial preparation were used for these observations. In four of the animals, under both general (ketamine hydrochloride, 40 mgkg) and local (0.5 ml 2% lignocaine with 1:100,000 epinephrine) anesthesia the dental pulp was amputated from the lower canine teeth on one side by removing 2-3 mm of the crown tip and debriding the root canal to the apical constriction with endodontic files and 2% sodium hypochlorite solution. The canal space was filled with gutta percha and zinc oxide and eugenol cement. The fifth animal acted as a n unoperated control, in which the teeth were left intact. Three months after pulpectomy the animals were killed by intravasuclar perfusion of ice-cold 2% glutaraldehyde and 0.1%osmium tetroxide in 0.01 M cacodylate buffer after initial clearing of the circulation with 0.01 M cacodylate buffer to which heparin and procaine hydrochloride had been added. Perfusion was maintained for 15 min. Mandibles from three animals were decalcified in formic acid. The canine teeth and supporting bone from these animals were embedded in paraffin for light microscope examination. Two mandibles were decalcified in 4% EDTA containing 1%glutaraldehyde a t 4°C. The decalcification was monitored radiographically and when it was complete, the region of the mandible containing the root of the canine tooth was slit into 1-mm-thick slices, the radiographs being used to control orientation and ensure that the periapical tissues were included. These slices were then processed for electron microscopy and embedded in LX112 resin. Received May 13, 1987; accepted July 23, 1987. PERIAPICAL INNERVATION AFTER PULPECTOMY Fig. 1. Periapical region immediately adjacent to the apical canal (A) entering the tooth pulp from a n unoperated tooth. C, cementum; B, bone; NF, nerve fibers. Hematoxylin and eosin. Fig. 2. Periapical region showing separate nerve fiber bundles (NF)passing to the periodontal ligament (upper) and to the apical canal of the tooth (lower). Unoperated tooth, siIver-stained. B, bone; C, cementum of root apex. 3 19 320 G.R. HOLLAND Fig. 3. Nerve fiber bundles (NF) in the periodontal ligament lateral to the apical foramen. B, bone; C , cementum. Unoperated tooth, silver-stained. Light Microscopy The paraffh blocks containing the canine roots and surrounding tissues were serially sectioned a t 8 pm. Every 10th section was stained with hemotoxylin and eosin. The region where the blood vessels and nerves entered the tooth pulp at the root apex was identified from those sections and 50 serial sections to each side of this central plane were stained with silver to demonstrate the innervation by the method described by Linder (1978). The hemotoxylin- and eosin-stained sections of the periapical region were examined for evidence of gross changes in the structure of the tissues. The silver-stained sections were used to construct three-dimensional reconstructions of the periapical innervation. Each section of the periapical series was examined in the photomicroscope and traced by means of a camera lucida attachment at a magnification of 200 X. Only the profile of the root apex and the silver-stained nerves were traced onto d e a r acrylic sheets. These were assembled into a reconstruction in two ways. Initially the sheets were merely manually superimposed, the profile of the root apex being used as a fixed point. A final composite reconstruction was made by adding a final sheet to the pile and tracing all the underlying nerve fibers onto it, effectively compressing the information from a 800-pm slice of tissue onto one sheet. In addition the nerve and apical profiles were digitized with software (Bioquant, R&M Biometrics, Nashville, TN) that would, as well as store the data, build the reconstruction and rotate it in various planes to determine whether the relationships we saw in single sections or in the superimposed series occurred in three dimensions. Electron Microscopy The plastic-embedded blocks were all initially sectioned at 1 pm and the first sections of each block were examined after toluidine blue staining. Regions of the block containing the subapical nerve fibers were located and the blocks were trimmed to include them only. These were then thin-sectioned, stained with uranyl acetate and lead citrate, and examined in the electron microscope. The innervation was located and examined and low-power montages were prepared of some components. Axon sizes were measured with a digitizing pad and appropriate software (Bioquant, R&M Biometrics, Nashville, TN). RESULTS Nerve Plexus in the Periapical Region In individual sections stained with toluidine blue or hemotoxylin and eosin the dental branches of the inferior alveolar nerve can be distinguished only with difficulty and little can be discerned of their distribution or arrangement except that they pass through clear channels in the bone and are almost always adjacent to blood vessels (Fig. 1). The use of a selective silver stain shows the neural arrangement more clearly. In single sections small seg- PERIAPICAL INNERVATION AFTER PULPECTOMY 321 appear widely separated compared with the inferior alveolar nerve and the endoneurium is sparse and poorly developed. Some smaller bundles containing only 2-5 myelinated axons also occur, as do occasional bundles containing only nonmyelinated axons. Occasional nonmyelinated fibers containing only one mitochondria-rich axon are seen close to the root apex. The bundles sometimes pass close to the bone, being separated from it only by a lining of osteoblasts. Immediately adjacent to the root tip the bundles are much smaller and appear equidistantly spaced from the cemental surface. A sampling of the nerve bundles shows that 45% of the axons in this area are nonmyelinated. The myelinated axons range in size from 4 to 38 pm in circumference. The size distribution is askewed unimodal with the mode at 15 pm (mean 17.13; SD 5.93); 35% of the myelinated axons are in the A/3 size range. Occasional lamellated axons were seen close to the tooth apex. Affect of Pulpectorny on the Periapical Plexus Fig. 4. Reconstruction of the periapical innervation made by superimposing camera lucida tracings from silver-stained sections. The hatched area represents the profile of the tooth apex. The larger bundle to the left is part of the inferior alveolar nerve trunk. Two large but separate branches innervate the tooth pulp and the periodontal ligament around the tooth apex. The plexus around the apex is contained within the periodontal ligament. Unoperated tooth. The principal difference in operated specimens is the presence of areas of inflammation in the subapical tissues (Fig. 7). In some sections it appears that the periodontal space innervation is sparse (Fig. 8), and in some sections it appears that branching is much more extensive in the bone between the inferior alveolar nerve trunk and the tooth apex. The manually superimposed compound images confirm these superficial impressions (Fig. 9). Only occasional bundles are found in the periodontal space of the apex. The branching pattern in the bone is more extensive, resulting in a larger number of more randomly arranged small branches. The computer reconstruction (Fig. 10) shows the changes even more clearly. As the image is rotated through O", 40°,80°,and 120" the presence of a proliferated mass of nerves in the bone beneath the apex is confirmed as is the almost total loss of the periodontal plexus, those fibers that remain being largely a greater distance from the cemental surface than in the control apices. The tissue is infiltrated with chronic inflammatory cells including lymphocytes and macrophages. The nerve bundles appear structurally normal even within the inflamed zone. Inflammatory cells do not appear within the epineurium and there is no overt evidence of neural degeneration. The structural characteristics noted for the control side were maintained on the operated side. A higher proportion of the axons were nonmyelinated (59%)and these axons showed a somewhat higher incidence of axoaxonal apposition and exposure of the axolemma to the extracellular space. The size range and distribution of the myelinated axons were similar to those of the control side. The mean size of myelinated axons was 18.17 pm (SD 7.711, not statistically significantly different from that on the control side (MannWhitney, P < 0.01). The occasional lamellated receptor terminal was also present in the operated as in the control pericemental zone. ments of the bundles traveling towards the apex may be seen (Fig. 2), as well as individual bundles in the periodontal space (Fig. 3). The images produced by manually superimposing serial tracings give a clearer presentation of the branching of these nerve bundles (Fig. 4).A large trunk leaves the inferior alveolar nerve and divides dichotomously several times before reaching the root apex. The daughter branches continue in the same general direction, ending in different segments of the apical periodontal space. Once approximated to the root apex some branches enter the apical foramen, but the majority end in the periodontal space by branching extensively and forming a definite neural plexus around the root apex midway between the cemental and bone surfaces. The examination of individual sections (Fig. 5) shows that in this periodontal plexus most fibers are oriented parallel to the cemental surface and most accompany arterioles or capillaries. Examination of the computer-generated reconstructions (Fig. 6) allows extension of these observations into three dimensions. At the 0" rotation (around the Y axis) (Fig. 6A) the density of the periodontal plexus can be seen, its relationship parallel to the root surface and the larger bundles that supply its branches. Rotating the image through 40" (Fig. 6B), 80" (Fig. 6 0 , and 120" (Fig. 6D) shows that the relationships that were seen in two dimensions persist in the third dimension and that the periodontal plexus is indeed a three-dimensional network completely enveloping the root apex on all sides. The nerve bundles derived from the inferior alveolar DISCUSSION nerve are relatively small when found in the immediThe normal arrangement of the innervation of the ately subapical zone. Each bundle contains 10-30 myelinated axons accompanied by a similar number of periapical tissues of the ferret consists of mixed myelinnonmyelinated axons. The axons within the bundles ated and nonmyelinated fibers in small bundles derived - 322 G.R. HOLLAND Fig. 5. A segment of the periodontal ligament (PDL) between cementum (C) and bone (B). Nerve fibers (arrow) are restricted largely to the central region of the ligament adjacent to blood vessels. Unoperated tooth, silver-stained. from the inferior alveolar nerve. The bundles branch dichotomously within the bone between the nerve trunk and the tooth apex. Some bundles travel directly to the root apex and, while giving some branches to the periodontal space, enter the apical foraminae to supply the dental pulp. Other bundles divert from the most direct root to the apex and pass through bone to reach the periodontal space more coronally. There is little branching within the bone but once the bundles reach the periodontal space extensive division occurs to form a network of smaller bundles and individual fibers, a distinct periodontal plexus. Three-dimensional reconstruction emphasizes the density of this plexus within a relatively confined but well-defined area. It appears to surround the apex on all sides. The plexus is almost entirely limited to the middle third of the ligament space with relatively nerve-free zones between it and both the cementum and bone. The nerve bundles almost always accompany blood vessels. An initial impression is that this plexus is much denser in the area immediately around the apex than a t more coronal levels though this point requires further investigation. This description corresponds with those by Byers (1985) of the rat molar and by Kubota and Osanai (1977) of the Japanese shrew mole. The role of the periodontal innervation has been subject to some debate (Hannam, 1985).While the response properties of individual fibers have been described in some detail (Hannam, 19691, the activity of this input in reflex and volitional jaw movements is unclear. This study can add little to the debate on function but such a n extensive plexus, precisely positioned in the periodontal space, must provide during mastication a substantial input on both tension and compression. The proximity of nerves and blood vessels is interesting. Presumably the blood vessels are placed such that their patency is maintained during tooth movement. This would, one would suppose, be the position of minimum stress and not the optimum position for receptors. It may be that the terminals as opposed to the fibers are positioned in more appropriate sites. This requires further study. The composition of the nerve bundles as they approach the apex differs from the composition of the inferior alveolar nerve (IAN) (Holland, 1978; Fried and Hildebrand, 1982) in that, unlike the inferior alveolar nerve trunk, the myelinated spectrum is unimodal and from the intrapulpal innervation (Holland and Robinson, 1983; Johnsen and Karlsson, 1974; Fried and Hildebrand, 1981) in the higher proportion of AP fibers. The proportion of non-myelinated axons is similar to that of the IAN and much smaller than that of the pulp. The high proportion of axoaxonal exposure reported in pulpal nerves (Holland, 1981) is absent. PERIAPICAL INNERVATION AFTER PULPECTOMY \ , v::.,.>:.:.:.>>:: :.>:.>:.. ........ ......... ............... ........ I 6A v....... ::::::. ......... :.:.:.:.:.>:. 323 ......> ...:.>:1 .......... ............. 2mm 4 6C u 2mm 6D Fig. 6. Computer reconstruction of the periapical innervation of a control tooth rotated around the Y axis. The profile of the tooth apex has been shaded for clarity. Rotation are as follows. A 0". B: 40". C: 80". D: 120". The relationship of the nerve fibers in the periodontal ligament to the root surface remains relatively constant, suggesting it is a three-dimensional plexus present on all sides of the root surface. The nerve bundles supplying the periapical tissues contain both pulpal and periodontal fibers. The difference in composition between them and the pulpal nerves would suggest that the bulk of the AP fibers go to the periodontal tissues, though within the ligament the fiber composition is as yet unknown. This would correlate well with the known mechanoreceptive role of the periodontal ligament and the nociceptive function of the dental pulp. The even higher proportion of A@fibers in the inferior alveolar nerve trunk would presumably be an expression of the cutaneous and mucosal fibers it contains, which are absent from the periapical bundles. It seems likely too that a larger proportion of the nonmyelinated axons enter the dental pulp than the periodontal ligament. This study did not include a detailed examination of the receptors or nerve terminals though we did observe both mitochondria-rich axonal segments and lamellated Paccinian corpuscle-like endings. If structure reflects function as elsewhere (Sakada, 1971), it would suggest that these lamellated bodies are highfrequency, fast-adapting transducers and would probably signal the initial onset of tooth movement. 324 G.R. HOLLAND Fig. 7. Nerve fibers passing through an area of subapical inflammation beneath a pulpectomized tooth. The infiltrate of leukocytes suggests a mild degree of inflammation. No damage to the nerve bundle is apparent. Hematoxylin and eosin. B, bone; C , cementum; NF, nerve fiber bundles. Fig. 8. Segment of periodontal ligament (PDL) from a pulpectomized specimen. No nerve fibers are present in this area. Silver stain (no counterstain). B, Bone. 325 PERIAPICAL INNERVATION AFTER PULPECTOMY 1m m Fig. 9. Reconstruction of the periapical innervation made by superimposing camera lucida tracings from silver-stained sections. The hatched area represents the profile of the tooth apex. The large bundle to the right is part of the inferior alveolar nerve. Most of the periodontal plexus is absent. The accumulation of small plexus nerve fibers in the subapical region is more extensive than in unoperated specimens. Pulpectomy has a dramatic effect on the innervation of the periapical region when observed 3 months later. It was somewhat surprising to find residual inflammation at this time. The degree of inflammation did not cause smicient bone loss to be detectable radiographically and consists of a leukocytic infiltration with little overt evidence of destruction. Within inflammed areas in the pulp, the nerves are the last elements to be affected (Torneck, 1974).There were no inflammatory cells within the perineurium and the structure of most of the myelinated axons appeared normal. The nonmyelinated axons, however, commonly. showed degenerative changes. Presumably the protection afforded the unmyelinated axons from extracellular toxins is not as effective as the multiple layered sheath of the myelinated axons. What is seen a t this 3-month stage is probably partial recovery from the acute trauma. It may be that observations at earlier stages (currently under way) and later (planned) will show a different pattern, perhaps more extensive damage initially and maybe a more normal pattern finally. If this is so, the repair process would appear to be extended compared to that observed after simple transverse section of a nerve trunk in this region (Holland and Robinson, 19851, where after similar time periods the peripheral innervation had almost entirely recovered. The composition of subapical bundles differed little from controls in terms of myelinated axons. A somewhat higher proportion of nonmyelinated axons after pulpec- J c 10B ./ Fig. 10. Computer reconstruction of the periapical innervation of a pulpectomized tooth rotated around the Y axis. The profile of the tooth apex has been shaded. The Rotation is as follows. A 0". B: 40".C: 80'; D: 120". By comparison with the control specimens (Fig. 6 ) it appears that the periodontal plexus has virtually disappeared and a network of small nerve fibers between the tooth apex and the nerve trunk is present. i G.R. HOLLAND 326 2mm 1oc .* Fig. 10, continued. tomy may indicate that the reparative process still is under way. The major change discernible, particularly in the three-dimentional reconstructions is the loss of the periodontal plexus in the ligament and the increased branching and accumulation of nerves below the apex but outside the ligament. If this change were to persist long, it may explain the differences in sensory discrimination between vital and nonvital teeth as being due to a loss of periodontal rather than pulpal input. While it would be attractive to consider the subapical neural reaction to be a neuroma, examination of this ultrastructure denies this. It appears merely to be a proliferation and increased branching with less confusion than demonstrated in neuromas (Blumberg and Janig, 1982). It may well later organize and from it branches may return t o the periodontal ligament and reform the plexus. At 3 months more extensive recovery may have been expected. After similar periods of time we have seen pulp and dentin reinnervated from nerve section lesions some distance away (Holland, Matthews and Robinson, 1987). It may be that severed pulpal nerve attempts to recover and reinnervate the target organ that has gone and that this reorganization has a longer time course than that occurring after simple transection of a nerve trunk. ACKNOWLEDGMENTS This study was supported by the Medical Research Council of Canada and the Alberta Heritage Foundation for Medical Research. I would like to thank Mrs. E. Pehowich and Mrs. V. Burgess for their expert technical assistance and Mrs. H. Ridyard for typing the manuscript. LITERATURE CITED Anderson, D.J., A.G. Hannam, and B. Matthews (1970) Sensory mechanisms in mammalian teeth and their supporting structures. Physiol. Rev., 50:171-195. Beasley, W.L., and G.R. Holland (1978)A quantitative analysis of the innervation of the pulp .~ of the cat’s canine tooth. J. Comp. Neurol., 178:487-494. Blumberg, H., and H. Janig (1982) Changes in unmyelinated fibers including postganglionic fibers of a shin nerve after peripheral neuroma formation. J. Auton. Nerv. Syst., 6:173-183. Burchiel, K.J., and L.C. Russell (1985)Spontaneous activities of ventral root axons following peripheral nerve injury. J. Neurosurg., 62:408-413. Byers, M.R. (1985) Sensory innervation of periodontal ligament of rat molars consists of unencapsulated Riffini-like-mechano-receptors and free nerve endings. J. Comp. Neurol., 231500-518. Cadden, S.W., S.W.J., Lisney, and B. Matthews (1982)A fibre innervation of tooth pulp in the cat, with a discussion of the functions of nerves supplying tooth-pulp. In: Anatomical, Physiological and Pharmacological Aspects of Trigeminal Pain. B. Matthews and R.G. Hill, eds. Excerpta Medica: Amsterdam, pp. 41-50. Fried, K., and C. Hildebrand (1981)Pulpal axons in developing, mature and ageing feline permanents incisors. J. Comp. Neurol., 203:3751. Fried, K., and C. 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