The ultrastructure of ruffini endings in the periodontal ligament of rat incisors with special reference to the terminal schwann cells (K-cells).код для вставкиСкачать
THE ANATOMICAL RECORD 223:95-103 (1989) The Ultrastructure of Ruffini Endings in the Periodontal Ligament of Rat Incisors With Special Reference to the Terminal Schwann Cells (K-Cells) TAKEYASU MAEDA, OSAMU SATO, SHIGEO KOBAYASHI, TOSHIHIKO IWANAGA, AND TSUNEO FUJITA Department of Oral Anatomy, Niigata University School of Dentistry, Gakkocho (TM., O.S., S.K.); and Department ofAnatomy, Niigata University School of Medicine, Asahimachi (TI., Ill?), Niigata 951, Japan ABSTRACT The Ruffini endings and associated cells in the periodontal ligament of rat incisors were investigated by means of immunohistochemistry for glia-specific S-100 protein and electron microscopy. Numerous Ruffini endings, which were immunoreactive for S-100 protein as well as for neurofilament protein, were distributed in the alveolus-related part of the lingual periodontal ligament. In electron microscopy, the Ruffini endings displayed expanded axoplasmic spines filled with a large number of mitochondria and neurofilaments; some of the spines directly contacted the surrounding collagen fibers via fingerlike projections. The axoplasmic spines and Schwann sheath, for the most part, were covered alternately by single or multiple layers of the basal lamina. Several rounded cells showing S-100 immunoreactivity occurred in the vicinity of the Ruffini endings. The rounded cells associated with Ruffhi endings possessed a kidney-shaped nucleus and enveloped the axoplasmic spines with their cytoplasmic processes. From these morphological features, the cells in question were identified a s the K-cells described by Everts et al. (1977). These K-cells developed Golgi apparatus and rough endoplasmic reticulum, suggesting active synthesis of proteins. Immunohistochemistry at the electron microscopic level revealed a n intense immunoreactivity for S-100 protein in the cytoplasm of the K-cell and led to a conclusion that the K-cells were terminal Schwann cells associated with R e i n i endings, presumably corresponding to the lamellar cells in the inner bulb of sensory corpuscles. Physiological studies and clinical experience have shown that the mechanical stimuli to the periodontal ligament induce oral reflexes, including the jaw-opening and closing reflex and also that these oral reflexes make regular and smooth mastication possible (Matthews, 1975). Nerves distributed in the dental pulp are responsible for pain sensation, whereas those in the periodontal ligament transmit both this and the sense of touch, pressure, and displacement in the teeth. The periodontal ligament has been shown physiologically to contain two types of mechanoreceptors: slowly and rapidly adapting receptors (Hannam, 1982). It has been suggested that the slowly adapting receptors respond to the stretch of periodontal fibers (Cash and Linden, 1982). By means of immunohistochemistry for neurofilament protein (NFP), our research group recently succeeded in demonstrating, in the periodontal ligament of rat molars and incisors, peculiar Ruffini-like nerve endings believed to function as stretch receptors (Maeda et al., 1987; Sat0 et al., 1988). In particular, a characteristic distribution of NFP-positive Ruffini endings was revealed in the rat incisors by Sat0 et al. (1988): Numerous Ruffini endings gathered in the midregion of the lingual periodontal ligament, whereas the labial periodontal lig0 1989 ALAN R. LISS. INC ament contained only free nerve endings. The Ruffini endings were found to be closely related to transverse periodontal fibers that directly connect the tooth with the alveolar bone. Such a distribution of endings seemed purposeful for the mechanoreception in the periodontal ligament, because the lingual periodontal ligament in rodent incisors is regularly stretched during mastication. While observing Ruffini endings in the periodontal ligament of rat incisors under the electron microscope, peculiar cells were found, which were associated with the nerve terminals. These cells appeared to correspond to the cells originally described by Beertsen et al. (1974) and more precisely studied by Everts et al. (1977). The latter authors recognized rounded cells closely associated with nerve terminals in the periodontal ligament of mice incisors and termed them K-cells because of their "kidney-shaped" nucleus. However, the origin and functional significance of these cells remains to be elucidated. Received March 21, 1988; accepted June 10, 1988. Address reprint requests to Takeyasu Maeda, Department of Oral Anatomy, Niigata University School of Dentistry, Gakkocho 2, Niigata 951, Japan. 96 T. MAEDA ET AL. Fig. 1. The lingual periodontal ligament of the rat incisor. PAP staining with S 100-antiserum. S-100 immunopositive neural elements ramify in a dendritic fashion and form Ruffni endings (arrows), restricted to the alveolus-related part (AR). TR:tooth-related part. x280. Fig. 2. High magnification of Ruffhi endings immunostained for S100 protein. Three rounded cells associated with the endings are stained positively. X1,500. The purpose of the present study was to reveal the ultrastructural features of Ruffini endings in the periodontal ligament of rat incisors, with special reference to the K-cells. In order to reveal the cellular origin of the K-cells, we performed immunohistochemistry a t the light and electron microscopic levels for S-100 protein, dia-suecific uroteins. one of the established . solution for 3 days a t 4"C,a s previously reported (Maeda et al., 1986).After immersion in a 30% sucrose solution overnight, the specimens were rapidly frozen in dry iceacetone. Frozen sections, 20-40 pm thick, were cut with a freezing microtome. MATERIALS AND METHODS Animals Five male Wistar rats, weighing about 150-200 g, were used. The animals were anesthetized with a n intraperitoneal injection of pentobarbiturate (0.4 mlkg) and perfused with Bouin's fluid via the ascending aorta. The maxillae were removed immediately and immersed en bloc in the same fixative for 6 hours. Following fixation, the maxillae were decalcified with Plank-Rychlo's AR AS AX BL F K PL S Abbreviations alveolus-related part axoplasmic spines axon basal lamina fibroblast K-cells periodontal ligament sheath Y lmmunohistochemistry for 9100 Protein Floating sections were processed for the peroxidaseantiperoxidase (PAP) method according to Sternberger (1979) and incubated overnight a t 4°C with a n S-100 antiserum diluted 1:1,500. The S-100 antiserum was obtained from a rabbit injected with a n S-100 antigen purified from bovine cerebra. A detailed characterization of this antiserum has been reported previously (Masuda et al., 1983). To check the specificity of the immunoreactions, a n absorption test was performed using the antiserum pretreated with the antigen (10 pg/ml diluted antiserum). The antigen-absorbed antiserum did not stain any cellular or intercellular elements. Conventional and lmmunohistochemical Electron Microscopy An additional six rats were perfused with either 2.5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) or, for immunohistochemistry, a mixture of 4% paraformaldehyde and 0.5% glutaraldehyde in 0.05 M phosphate buffer (pH 7.4). The upper incisors then were removed and immersed in the same fixative for 6 hours. After RUFFINI ENDINGS IN RAT PERIODONTAL LIGAMENT 97 RESULTS lmmunohistochemistry for S-100 protein fixation, the specimens were decalcified in 4.13% EDTA2Na (ethylene diamine tetra acetic acid, disodium salt) solution for 3 weeks at room temperature. Decalcified tissue blocks were rinsed thoroughly overnight in the same buffer solution and cut sagittally at a 500 pm thickness with a vibratome (Oxford Co. Ltd.). For conventional electron microscopy, the sections were dehydrated after postfixation in 1% osmium tetraoxide for 3 hours, through a graded series of ethanol and embedded in Epon-812. Ultrathin sections were stained with both uranyl acetate and lead citrate and examined with a JEM-100 S X transmission electron microscope. Materials fixed with a mixture of paraformaldehyde and glutaraldehyde were used for immunohistochemistry. The vibratome sections were stained with the PAP method using the S-100 antiserum and mounted on polyL-lysine-coated glass slides. They were postfixed in 1% osmium tetroxide for 1hour and embedded in Epon-812. Ultrathin sections were observed in a transmission electron microscope without electron staining. The S-100 antiserum could demonstrate clearly the courses of nerves in the periodontal ligament of rat incisors, as previously reported (Sat0 et al., 1988),in the sections fixed with both Bouin’s fluid and a mixture of paraformaldehyde and glutaraldehyde. Dense concentrations of Ruffini endings showing S-100 immunoreactivity were found in the midregion of the lingual periodontal ligament. Ruffini endings were restricted to the alveolar half of the periodontal ligament, which was referred to a s the alveolus-related part (Fig. 1).Observation of the cross-sectioned endings indicated that the Schwann sheath surrounding axon terminals was selectively immunostained with the S-100 antiserum; this finding was confirmed by immunohistochemistry at the electron microscopic levels. Rounded cell bodies showing intense immunoreactivity for S-100 protein frequently occurred near the axo- Fig. 3.Electron micrograph showing an axoplasmic spine (AS) and (K) in the collagen-rich zone of the periodontal ligament (PL). K-cells (terminal Schwann cells) possess developed Golgi appara- tus (arrowheads) and broad endoplasmic reticulum (arrows). The cytoplasmic process of the K-cell envelopes the nerve terminals filled with a large number of mitochondria. ~ 9 , 3 0 0 . two K-cells 98 T. MAEDA ET AL. plasmic spines of the Ruffini endings. They were rich in cytoplasm and possessed a rounded or ovoid nucleus, frequently showing a marked indentation, thus taking a kidney-like shape (Fig. 2). Electron Microscopic Observation of Ruffini Endings Ruffhi endings in the lingual periodontal ligament were observed by electron microscope. They were easily identified as expanded axoplasmic spines filled with mitochondria. The axoplasmic spines were distributed exclusively within the collagen-rich zone. The axoplasmic spines contained a large number of neurofilaments and microtubules as well as mitochondria, but no synaptic vesicles or multivesicular bodies. The peculiar cells with the kidney-shaped nucleus (Kcells) were frequently recognized in the vicinity of the expanded axoplasmic spines (Fig. 3). Their rich cytoplasm contained well-developed Golgi apparatus and rough endoplasmic reticulum. The latter was characterized by broad intracisternal spaces that were more electron-dense than the cytoplasm elsewhere. The cytoplasmic process of the K-cells usually extended to axoplasmic spines to envelope them (Fig. 3). Furthermore, it was not uncommon for the broad cytoplasmic process of K-cells to surround circumferentially the axons filled with mitochondria or for one K-cell to hold more than two mitochondria-rich axons (Figs. 4,5). These findings lead us to regard the K-cells as terminal Schwann cells. The terminal Schwann cells developed numerous caveolae on the cell membrane facing the axoplasmic spines, whereas only a few caveolae were present on the other cell surface (Figs. 6-8). No desmosomal junctions were observed between the terminal Schwann cells and axoplasmic spines of the Ruffini endings. The sheath of terminal Schwann cells was not continuous, so that axoplasmic spines were partially exposed to the surrounding basal lamina or collagen fibers through the slits of the Schwann sheath (Figs. 6-9). The exposed portions of the axons frequently were equipped with fingerlike projections, with longer ones penetrating into collagen bundles a t right angles (Fig. 7b). The axoplasmic spines of Ruffini endings, together with their sheaths of terminal Schwann cells, were covered alternately by either single layer or multiple layers of basal lamina; the multiple layers of the basal lamina, which usually were circumferential, were not formed evenly between the Schwann covering and collagen fibers (Figs. 6, 7a, 9). The axoplasmic spines partially lacked the basal lamina, especially in their tip equipped with fingerlike projections (Fig. 7b). Moreover, surrounding fibroblasts devoted any basal lamina covering to the periodontal ligament of rat incisor. When we observed specimens immunostained for S100 by electron microscope, terminal Schwann cells pos- Figs. 4, 5. Electron microaaphs of K-cells (K) in the periodontal organelles. They embrace the mitochondria-rich axons (arrowheads) in ligament of a rat incisor. The K-cells (terminal Schwann cells) are their cytoplasmic extension. In Figure 5, the cytoplasmic process surcharacterized by their kidney-shaped nucleus and well-developed cell rounds circumferentially one axon. X 7,000. RUFFINI ENDINGS IN RAT PERIODONTAL LIGAMENT Fig. 6.An electron micrograph of branched nerve terminal in the periodontal ligament. Several fingerlike projections (arrows) extend through the slits of the Schwann sheath. Caveolae of Schwann sheath 99 (arrowheads) are more numerous on the side facing the axoplasmic spine than on the other surface. ~ 6 , 5 0 0 . vitz and Shore, 19781, and in the molars of rats (Byers, 1985), although some researchers did not consider the endings they observed to be Ruffhi endings. Byers (1985) described in detail the morphology of the Ruffini-like endings in the periodontal ligament of rat molars with the use of autoradiography and electron microscopy. The ultrastructural features of the Ruffini endings observed in the present study are virtually the same as those described by Byers (1985). It is a common feature that the axoplasmic spines sometimes extend fingerlike projections into the collagen zone, and these were completely free, with no Schwann cell or basal lamina covering. Byers (1985)reported that in rat molars there DISCUSSION is symmetric multiple layers of basal lamina constantly The ultrastructure of the Ruffini endings in the perio- forming between the Schwann sheath and collagen, aldontal ligament has been observed in the incisors of though this coverage of the basal lamina was incomplete mice (Evzrts et al., 1977), in the incisors of rats (Berko- in the periodontal ligament of incisors. sessing kidney-shaped nucleus were easily detected in the periodontal ligament because of their electron-dense reaction product (Fig. lob). The immunoreactive product for S-100 protein was distributed throughout the cytoplasmic matrix, but no reaction-or a weak reaction product-was present in the karyoplasm. Their cytoplasm contained well-developed, rough endoplasmic reticulum and mitochondria that were free from the S-100like immunoreactivity. On the other hand, the fibroblasts distributed in the periodontal ligament were negative in S-100 immunoreaction. 100 T. MAEDA ET AL. Fig. 7. Two axoplasmic spines (AS) in the collagen-rich zone of the periodontal ligament (PL) covered by the cellular sheath, possibly derived from that of the K-cell (terminal Schwann cell) and partially by several layers of basal lamina (EL). At the tip of the axoplasmic spine (rectangle, a), fingerlike projections directly contact the collagen fibers at right angles (arrowheads, b). a, ~ 5 , 0 0 0b, : ~16,000. K-cells could be recognized in association with the Ruffini endings in the periodontal ligament of rat incisors. Some researchers have reported these peculiar cells in the periodontal ligament of the rat (Beertsen et al., 1974; Berkovitz and Shore, 1978) and mouse incisors (Everts et al., 1977). These authors pointed out that the K-cells were closely associated with the terminal region of nerve fibers. Berkovitz and Shore (1978) also noticed the existence of nontypical Schwann cells surrounding the mitochondria-rich axons in the rat incisors. The present study confirmed the close relation of the K-cells to the Ruffini endings; the cytoplasmic process of K-cells directly enveloped the axoplasmic spines. These findings have led us to propose that the K-cells represent terminal Schwann cells. This idea is strongly supported by the immunohistochemical staining in which these cells were selectively immunoreactive for glia-specific S-100protein. Terminal Schwann cells were found to have numerous caveolae on the cell membrane, this being a morphological feature in common with the lamellar cells of Meissner’s and Pacinian corpuscles, which were immunoreactive for S-100 protein (Iwanaga et al., 1982). In the present study, we observed some terminal Schwann cells circumferentially embracing the mitochondria-rich asoplasmic spines with their cell body or cell process. This figure may represent a transitional form into the lamellated corpuscles such as Pacinian and Meissner’s corpuscles. Berkovitz et al. (1983) found one lamellated nerve corpuscle in the periodontal ligament of rat incisors, although other researchers (Sato et al., 1988) including the present study, were unable to observe such lamellated corpuscle in the same tissue. It is notewnrthy that terminal Schwann cells in the periodontal ligament contained well-developed cell organella, especially the Golgi apparatus and rough endoplasmic reticulum. These cytological features are Fig. 8. An electron micrograph of a Ruffini ending, in the lingual periodontal ligament (PL) of the rat incisor. An axoplasmic spine (AS) containing a large number of mitochondria is surrounded by the sheath of K-cell (terminal Schwann cell). This cellular sheath is not continuous, so that the axoplasmic spine frequently is exposed to the surrounding collagen fibers (arrows). x 12,000. generally related to a n active synthesis of proteins. Highenzyme activities, including nonspecific cholinesterase, were demonstrated to be localized a t the Golgi apparatus and rough endoplasmic reticulum in the lamellar cells of Pacinian corpuscles (Ide and Saito, 1980) and cat Ruffini endings (Toyoma, 1985). In our preliminary study, terminal Schwann cells in the periodontal ligaments of rat incisors were shown to contain intense cholinesterase activity in the endoplasmic reticulum (authors’ unpublished observation). A clear enzyme activity for cholinesterase also was localized in the caveolae of the terminal Schwann cells, which predominantly were found on the cell membrane facing the axoplasmic spines in Ruffini endings. These findings suggest that the caveolae in the terminal Schwann cells might be involved in conveying these enzymes and other substances. The possibility arises that these terminal Schwann cells may differentiate to lamellar cells of the sensory corpuscles. Fig. 9. A cross-sectioned axoplasmic spine (AS) of a Ruffini ending. The axoplasmic spine lacks both the glial sheath and multiple layer of basal lamina (BL) on the lateral side and directly contacts the collagen fibers with fingerlike projections (arrows). ~ 9 , 0 0 0 . 102 T. MAEDA ET AL. Fig. 10. Immunohistochemistry for 5-100 protein at electron microscopic level. The Schwann cell and its sheath is) are specificab immunostained with S-100 antiserum. a: The axon and myelin sheath (AX) are negative in reaction. ~ 6 , 0 0 0b: . The cytoplasm of the K-cells (K) is filled with electron-dense immunoreacted materials. On the other hand, the adjacent fibroblast (F)is free from the S-100 immuno- Little information is available on the functional significance of terminal Schwann cells in the periodontal Ruffini endings. The existence of cells resembling periodontal terminal Schwann cells has not been reported in the Ruffini endings of other tissues. Moreover, Byers (1985) failed to observe the terminal Schwann cells associated with the Ruffini-like endings in the periodontal ligament of rat molars. The functions of the terminal Schwann cells possibly may be related to some condition characteristic of the rodent incisor. As the incisors of rodents grow continuously throughout life, active remodeling of the periodontal fibers should take place there. These terminal Schwann cells may have a special function in extending support and nutrition to nerve terminals under such severe conditions. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (No. 63440069 and 63790412). ACKNOWLEDGMENTS The authors are grateful to Professor Y. Takahashi, Department of Neuropharmacology, Brain Research Institute, Niigata University, for his generous gift of the antiserum and antigen against S-100 protein. We also thank Mr. M. Hoshino for his photographic assistance. x8,000. LITERATURE CITED Beertsen W., V. Everts, and A. Van den Hooff 1974 Fine structure and possible function of cells containing leptomeric organelles in the periodontal ligament of the rat incisor. Arch. Oral Biol., 19t10991100. Berkovitz, B.K.B., and R.C. Shore 1978 High mitochondria1 density within peripheral nerve fibers of the periodontal ligament of the rat incisor. Arch. Oral Biol., 23t207-213. Berkovitz, B.K.B., R.C. Shore, and B.J. Moxham 1983 The occurrence of a lamellated nerve terminal in the periodontal ligament of the rat incisor. Arch. Oral Biol., 28.99-101. Bycrs, M.R. 1985 Sensory innervation of periodontal ligament of rat molars consists of unencapsulated Ruffini-like mechanoreceptors and free nerve endings. J. Comp. Neurol., 231t500-518. Cash, R.M., and R.W.A. Linden 1982 The distribution of mechanoreceptors in the periodontal ligament of the mandibular canine tooth of the cat. J. Physiol., 330:439-447. RUFFINI ENDINGS IN RAT PERIODONTAL LIGAMENT Everts, V., W. Beertsen, and A. Van den Hooff 1977 Fine structure of a n end organ in the periodontal ligament of the mouse incisor. Anat. Rec., 189t73-90. Hannam, A.G. 1982 The innervation of the periodontal ligament. In: The Periodontal Ligament in Health and Disease. B.K.B. Berkovitz, B.J. Moxham, and H.N. Newman, eds. Pergamon Press, Oxford, pp. 173-196. Ide, C., and T. Saitoh 1980 Electron microscopic histochemistry of cholinesterase activity of Vater-Pacini corpuscles. Acta Histochem. Cytochem., 13t298-305. Iwanaga, T., T. Fujita, Y. Takahashi, and T. Nakajima 1982 Meissner’s and Pacinian corpuscles as studied by immunohistochemistry for S-100 protein, neuron specific enolase and neurofilament protein. Neurosci. Lett., 31:117-121. Maeda, T., T. Iwanaga, T. Fujita, and S. Kobayashi 1986 Immunohistochemical demonstration of nerves in the predentin and dentin of human third molars with the use of an antiserum against neurofilament protein (NFP). Cell Tissue Res., 243t469-475. Maeda, T., T. Iwanaga, Y. Takahashi, T. Fujita, and S. Kobayashi 1987 103 Distribution of nerve fibers immunoreactive to neurofilament protein in rat molars and periodontium. Cell Tissue Res., 249:13-23. Masuda, T., K. Sakimura, Y. Yoshida, R. Kuwano, T. Isobe, T. Okuyama, and Y. Takahashi (1983) Developmental changes in the translatable mRNA for subunit of 5-100 protein in rat brain. Biochem. Biophys. Acta, 74Ot249-254. Matthews, B. 1975 Mastication. In: Applied Physiology of the Mouth. C.L.B. Lanelle, ed. Wrights, Bristol, pp. 199-242. Sato, O., T. Maeda, S. Kobayashi, T. Iwanaga, T. Fujita, and Y. Takahashi 1988 Innervation of periodontal ligament and dental pulp in rat incisors. An immunohistochemical investigation of neurofilament protein and glia-specific S-100 protein. Cell Tissue Res., 251t13-21. Sternberger, L.A. 1979 Immunohistochemistry. 2nd ed, Prentice-Hall, Inc., Englewood Cliffs, New Jersey. Toyoma, Y. 1985 The morphology of sensory corpuscles in joint capsule-Ultrastructural and histochemical study. J. Jpn. Orthop. Assac., 59r397-407.