ARTICLE |
Correspondence to: Teruko TakanoYamamoto, Dept. of Orthodontics, Okayama Univ. Dental School, 2-5-1, Shikata-cho, Okayama 700-8525, Japan. E-mail: t_yamamo@dent.okayama-u.ac.jp
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The periodontal ligament is the connective tissue that fills the space between the tooth and its bony socket. It is abundantly innervated by the sensory and sympathetic nerves. We first investigated the immunoreactivity of TrkA, which is a high-affinity receptor of nerve growth factor (NGF), in the periodontal ligament of rats. Immunoreactivity was observed at the epithelial cells in the cervical and furcation regions of the molars. These epithelial cells, which gather together to form clusters or networks, are known as the epithelial rests of Malassez. Immunoreactivity was not observed in other non-neuronal cells, such as osteoblasts, fibroblasts, odontoblasts, cementoblasts, endothelial cells, and/or osteoclasts. On the basis of these findings, we investigated the possible involvement of sensory nerve innervation in the immunoreactivity of the epithelial cells. Denervation of the inferior alveolar nerve resulted in a marked decrease in the distribution area and size of the clusters of immunoreactive cells compared with those of sham-operated rats. These findings suggest that sensory nerve innervation may have a regulatory role in maintenance of the epithelial rests of Malassez expressing TrkA in the periodontal ligament. (J Histochem Cytochem 48:979984, 2000)
Key Words: TrkA, epithelial rests of Malassez, sensory nerve innervation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The periodontal ligament is the connective tissue that fills the space between the tooth root and its bony socket (
Nerve growth factor (NGF) is essential in promoting the development, maintenance, and survival of sensory and sympathetic neurons (
Previous ultrastructural observation revealed an intimate relationship between the sensory nerve endings and the basal lamina of the epithelial rests of Malassez (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissues
Twenty 7-week-old male Wistar rats were used. The animals were maintained on a normal solid food diet and water given ad libitum. The rats were deeply anesthetized with sodium pentobarbital and transcardiacally perfused with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The mandibular alveolar bones were immediately excised, postfixed for 1 day in the same fixative as was used for perfusion, and decalcified in 10% ethylendiaminetetra-acetate (EDTA) solution for 14 days. Sagittal and horizontal sections 50 µm thick were cut on a cryostat (Sakura; Tokyo, Japan). Horizontal sections of 7 µm were also cut on a microtome (Leica; Oberkochen, Germany) after embedment in paraffin.
Immunohistochemistry for TrkA
Mandibles from 10 rats were used in this study. Serial sagittal and horizontal sections 50 µm thick were treated in the free-floating state through the labeling step. Serial horizontal sections of 7 µm were cut on a microtome after the decalcified bone had been embedded in paraffin. The sections were incubated with rabbit anti-TrkA antibody (sc-118; Santa Cruz Biotechnology, Santa Cruz, CA; 1:8000) at 4C for 48 hr. Antigenantibody complexes were detected by the avidinbiotinperoxidase (ABC) method (Vectastain ABC kit; Vector, Burlingame, CA). The specificity of the TrkA antibody (sc-118) had been previously proved (
Nerve Denervation
Ten rats were used for the denervation study. Denervated rats were prepared by inferior alveolar nerve transection (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunohistochemistry for TrkA
In the 50-µm sections, immunoreactivity indicating TrkA was observed only in the epithelial rests of Malassez in the cervical and furcation regions of the molars. No immunoreactivity was observed in the nerve fibers (Fig 1).
|
In the 7-µm sections, strongly positive TrkA immunoreactivity was observed at the cell surface of epithelial rests of Malassez in the periodontal ligament (Fig 2). The rests were localized close to the root surface. Immunoreactivity was not observed in other non-neuronal cells, such as osteoblasts, fibroblasts, odontblasts, cementoblasts, endothelial cells, and osteoclasts, or in neural tissues. These numerous epithelial cells showed oval or round cluster forms, and some cells showed a strand form (Fig 2B and Fig 2C). They were localized near the root surface rather than near the alveolar bone.
|
Influence of Denervation on Distribution of Immunoreactivity
To evaluate the possible association between sensory nerve denervation and expression of TrkA in epithelial rests of Malassez, we compared the distribution pattern of the immunoreactive cells in the furcation region of lower second molars between the specimens from sham-operated and denervated rats on 50-µm serial sections. Fig 3 shows a diagram of the location of the serial sections shown in Fig 4.
|
|
In the sham-operated rats (Fig 4A14A4), many TrkA-immunoreactive epithelial rests of Malassez were observed around the furcation regions of the teeth. They were arranged in strands or as networks of strands. In particular, epithelial cells were densely localized near the furcation (Fig 4A2 and 4A3).
In denervated rats, immunoreactivity was also observed in the epithelial rests of Malassez (Fig 4B14B4 and 4C14C4). However, 1 week after the denervation of inferior alveolar nerve, the size of the immunoreactive cell clusters around all the lower molars was smaller and their distribution area was narrower than those of sham-operated rats (Fig 4B14B4 and 5B). These tendencies were more prominent after 3 weeks, and immunoreactive epithelial cells became sparse (Fig 4C14C4 and 5C). These changes associated with denervation of the inferior alveolar nerve were observed in all the animals, i.e., 1-week-denervated (n = 5) and 3-week-denervated (n = 5) rats. The adjacent gingival epithelium showed intense TrkA immunoreactivity which was evaluated as a positive control for the immunoreactivity in the denervated specimens (not shown).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We found that TrkA immunoreactivity was observed in the epithelial rests of Malassez. They were situated close to the root surface in the horizontal sections and were localized in the cervical and furcation regions of the molars in the rat periodontal ligament in sagittal sections. These findings are in agreement with previous investigations on the distribution pattern of epithelial rests of Malassez (
TrkA is a membrane-bound protein and mediates the functional responses to NGF through activation of tyrosine kinase (
As seen in thin 7-µm sections, epithelial rests of Malassez showed islands of cluster forms, and the distribution pattern of these epithelial cells was in agreement with that found in a previous investigation (
The furcation regions of molars are regions at which the alveolar bone and periodontal ligament are remodeled actively, unlike any other regions around the tooth (
In the present study, it is noteworthy that inferior alveolar nerve transection caused remarkable changes in the pattern of distribution of TrkA-immunoreactive epithelial cells. Denervation decreased the number of the clusters or islands within 1 week, and they were further decreased in 3 weeks. Moreover, the size of the clusters or islands was also decreased. Transection of the sensory fibers leads to the degeneration of their peripheral portions, with separation from the neurons (
|
![]() |
Acknowledgments |
---|
Supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (10470446, 10557197, and 11771318).
Received for publication September 22, 1999; accepted March 6, 2000.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barbacid M (1994) The Trk family of neurotrophin receptors. J Neurobiol 25:1386-1403[Medline]
Beertsen W, McCulloch CA, Sodek J (1997) The periodontal ligament: a unique, multifunctional connective tissue. Periodontology 13:20-40[Medline]
Berkovitz BK, Whatling R, Barrett AW, Omar SS (1997) The structure of bovine periodontal ligament with special reference to the epithelial cell rests. J Periodontol 68:905-913[Medline]
Burette A, Jalenques I, Romand R (1997) Neurotrophin receptor immunostaining in the rat ventral cochlear nucleus. Brain Res 776:10-23[Medline]
Chao MV, Hempstead BL (1995) p75 and Trk: a two-receptor system. Trends Neurosci 18:321-326[Medline]
Christ GJ, Brink PR, Ramanan SV (1994) Dynamic gap junctional communication: a delimiting model for tissue responses. Biophys J 67:1335-1344[Abstract]
Fristad I (1997) Dental innervation: functions and plasticity after peripheral injury. Acta Odontol Scand 55:236-254[Medline]
Gao Z, Mackenzie IC, Williams DM, Cruchley AT, Leigh I, Lane EB (1988) Patterns of keratin-expression in rests of Malassez and periapical lesions. J Oral Pathol 17:178-185[Medline]
Garcia SO, Hannestad J, Esteban I, Martinez del Valle M, Naves FJ, Vega JA (1997) Neurotrophin receptor-like protein immunoreactivity in human lymph nodes. Anat Rec 249:226-232[Medline]
Heyeraas KJ, Kvinnsland I, Byers MR, Jacobsen EB (1993) Nerve fibers immunoreactive to protein gene product 9.5, calcitonin gene-related peptide, substance P, and neuropeptide Y in the dental pulp, periodontal ligament, and gingiva in cats. Acta Odontol Scand 51:207-221[Medline]
Kaplan DR, Hempstead BL, Martin ZD, Chao MV, Parada LF (1991) The trk proto-oncogene product: a signal transducing receptor for nerve growth factor. Science 252:554-558. [see Comments][Medline]
Kato J, Ichikawa H, Wakisaka S, Matsuo S, Sakuda M, Akai M (1990) The distribution of vasoactive intestinal polypeptides and calcitonin gene-related peptide in the periodontal ligament of mouse molar teeth. Arch Oral Biol 35:63-66[Medline]
Lambrichts I, Creemers J, Van SD (1993) Periodontal neural endings intimately relate to epithelial rests of Malassez in humans. A light and electron microscope study. J Anat 182:153-162[Medline]
LeviMontalcini R (1987) The nerve growth factor: thirty-five years later. EMBO J 6:1145-1154. [published erratum appears in EMBO J 1987 6:2856][Medline]
Lopez SM, Perez PM, Marquez JM, Naves FJ, Represa J, Vega JA (1998) p75 and TrkA neurotrophin receptors in human skin after spinal cord and peripheral nerve injury, with special reference to sensory corpuscles. Anat Rec 251:371-383[Medline]
Nemoto K, Fukamachi K, Nemoto F, Miyata S, Hamada M, Nakamura Y, Senba E, Ueyama T (1998) Gene expression of neurotrophins and their receptors in cultured rat vascular smooth muscle cells. Biochem Biophys Res Commun 245:284-288[Medline]
Parrens M, Labouyrie E, Groppi A, Dubus P, Carles D, Velly JF, de Mascarel A, Merlio JP (1998) Expression of NGF receptors in normal and pathological human thymus. J Neuroimmunol 85:11-21[Medline]
Shibayama E, Koizumi H (1996) Cellular localization of the Trk neurotrophin receptor family in human non-neuronal tissues. Am J Pathol 148:1807-1818[Abstract]
TakanoYamamoto T, Takemura T, Kitamura Y, Nomura S (1994) Site-specific expression of mRNAs for osteonectin, osteocalcin, and osteopontin revealed by in situ hybridization in rat periodontal ligament during physiological tooth movement. J Histochem Cytochem 42:885-896
Ten Cate A (1996) The role of epithelium in the development, structure and function of the tissues of tooth support. Oral Dis 2:55-62[Medline]
Vignery A, Baron R (1980) Dynamic histomorphometry of alveolar bone remodeling in the adult rat. Anat Rec 196:191-200[Medline]
Wakisaka S, Nishikawa S, Ichikawa H, Matsuo S, Takano Y, Akai M (1985) The distribution and origin of substance P-like immunoreactivity in the rat molar pulp and periodontal tissues. Arch Oral Biol 30:813-818[Medline]
Wesselink PR, Beertsen W (1993) The prevalence and distribution of rests of Malassez in the mouse molar and their possible role in repair and maintenance of the periodontal ligament. Arch Oral Biol 38:399-403[Medline]