S.H. Youn

The Ruffini Ending as the Primary Mechanoreceptor in the Periodontal Ligament: Its Morphology, Cytochemical Features, Regeneration, and Development

The periodontal ligament receives a rich sensory nerve supply and contains many nociceptors and mechanoreceptors. Although its various kinds of mechanoreceptors have been reported in the past, only recently have studies revealed that the Ruffini endings--categorized as low-threshold, slowly adapting, type II mechanoreceptors--are the primary mechanoreceptors in the periodontal ligament. The periodontal Ruffini endings display dendritic ramifications with expanded terminal buttons and, furthermore, are ultrastructurally characterized by expanded axon terminals filled with many mitochondria and by an association with terminal or lamellar Schwann cells. The axon terminals of the periodontal Ruffini endings have finger-like projections called axonal spines or microspikes, which extend into the surrounding tissue to detect the deformation of collagen fibers. The functional basis of the periodontal Ruffini endings has been analyzed by histochemical techniques. Histochemically, the axon terminals are reactive for cytochrome oxidase activity, and the terminal Schwann cells have both non-specific cholinesterase and acid phosphatase activity. On the other hand, many investigations have suggested that the Ruffini endings have a high potential for neuroplasticity. For example, immunoreactivity for p75-NGFR (low-affinity nerve growth factor receptor) and GAP-43 (growth-associated protein-43), both of which play important roles in nerve regeneration/development processes, have been reported in the periodontal Ruffini endings, even in adult animals (though these proteins are usually repressed or down-regulated in mature neurons). Furthermore, in experimental studies on nerve injury to the inferior alveolar nerve, the degeneration of Ruffini endings takes place immediately after nerve injury, with regeneration beginning from 3 to 5 days later, and the distribution and terminal morphology returning to almost normal at around 14 days. During regeneration, some regenerating Ruffini endings expressed neuropeptide Y, which is rarely observed in normal animals. On the other hand, the periodontal Ruffini endings show stage-specific configurations which are closely related to tooth eruption and the addition of occlusal forces to the tooth during postnatal development, suggesting that mechanical stimuli due to tooth eruption and occlusion are a prerequisite for the differentiation and maturation of the periodontal Ruffini endings. Further investigations are needed to clarify the involvement of growth factors in the molecular mechanisms of the development and regeneration processes of the Ruffini endings.

Regeneration of periodontal primary afferents of the rat incisor following injury of the inferior alveolar nerve with special reference to neuropeptide Y-like immunoreactive primary afferents.

Regeneration of primary afferents and the expression of neuropeptide Y (NPY) in the lingual periodontal ligament of the rat incisor were examined following different types of injury (resection or crush) of the inferior alveolar nerve (IAN) combined with superior cervical ganglionectomy. In normal animals, protein gene product 9.5 (PGP 9.5)-like immunoreactivity (-LI) was localized in the middle areas of the alveolus-related part of lingual periodontal ligament; some of these nerve fibers showed terminal ramification and morphologies resembling those of the periodontal Ruffini endings, and very few thin varicose NPY-like immunoreactive (-IR) nerve fibers were detected around the blood vessels. Three days following crush injury of the IAN, the number of PGP 9.5-IR nerve fibers decreased, then increased to the normal levels around 10-15 days following injury. NPY-IR primary afferents first appeared around 5 days following crush injury, increased in number gradually, reaching a peak around 14 days, and then decreased. No NPY-IR primary afferents were detected 56 days following crush injury of the IAN. The terminal morphology of NPY-IR primary afferents observed around 10-14 days following injury was similar to that of PGP 9.5-IR nerve fibers in the normal animals, but less expanded. The changes in distribution of PGP 9.5-IR and NPY-IR nerve fibers following resection were similar to those observed following crush injury but regeneration was slightly delayed. The present results suggest that injury-evoked NPY is closely associated with the regeneration process of mechanoreceptors in the periodontal ligament following injury of the IAN.

Protein gene-product 9.5 in developing mouse circumvallate papilla: comparison with neuron-specific enolase and calcitonin gene-related peptide

The present study was made to investigate the ontogeny of protein gene-product 9.5 (PGP 9.5)-like immunoreactivity (-LI) in the developing mouse circumvallate papilla (CVP), and its distribution was compared to that of neuron-specific enolase (NSE) and calcitonin gene-related peptide (CGRP). In adult CVP, PGP 9.5-LI was observed in the subgemmal nerve plexus; some thin PGP 9.5-like immunoreactive (-IR) nerve fibers penetrated taste buds and apical epithelium. PGP 9.5-LI was also observed in the spindle-shaped cells in taste buds, and a small number of round- or oval-shaped ganglionic cells in the lamina propria. The distribution of NSE-LI was comparable to that of PGP 9.5-LI. CGRP-LI was observed in the nerve fibers only; distribution of CGRP-IR nerve fibers was similar to that of PGP 9.5-IR nerve fibers, although the number of CGRP-IR nerve fibers was smaller than that of PGP 9.5-IR nerve fibers. At least six developmental stages were defined with regard to the developmental changes in the distribution of PGP 9.5-LI from embryonic day (E) 12 to adulthood: Stage I (E12–13) — a dense nerve plexus of PGP 9.5-IR nerve fibers was detected in the lamina propria beneath the core of newly-formed papilla. Stage II (E14–16) — thin PGP 9.5-IR nerve fibers penetrated the apical epithelium, and a few round-shaped cells in the apical epithelium also displayed PGP 9.5-LI. Stage III (E17–18) — thin PGP 9.5-IR nerve fibers penetrated the inner lateral epithelium of the trench. Stage IV [Postnatal day (P) 0–3] many PGP 9.5-IR nerve fibers penetrated the outer lateral epithelium of the trench; later in this stage, taste buds appeared. Stage V (P5–10) — a small number of PGP 9.5 IR cells in the taste buds appeared, and their number increased gradually. Stage VI (PI4-adult) — the number of PGP 9.5-IR taste cells increased and reached the adult level, while the number of PGP 9.5-IR nerve fibers decreased. The development of NSE-LI was similar to that of PGP 9.5-LI. CGRP-IR nerve fibers were detected at E12 in the lamina propria, and the development of the intraepithelial CGRP-IR nerve fibers was similar to that of PGP 9.5-IR nerve fibers. The present results indicate that invasion by nerve fibers of the epithelium of lingual papillae occurs in a complex manner, and that these nerve fibers may participate in the formation of the taste buds.

Neuropeptide Y-immunoreactive primary afferents in the dental pulp and periodontal ligament following nerve injury to the inferior alveolar nerve in the rat.

The distribution of neuropeptide Y (NPY)-immunoreactive (IR) primary afferents in the dental pulp and periodontal ligament of the rat mandible were examined following combined chronic constriction injury (CCI) of the inferior alveolar nerve (IAN) and sympathectomy of the superior cervical ganglion (SCG). NPY-IR nerve fibers were observed around the blood vessels in the trigeminal ganglion, dental pulp and periodontal ligament in normal animals. Following combined CCI of the IAN and sympathectomy of SCG (SCGx), perivascular NPY-IR nerve fibers originating from SCG disappeared completely, but many NPY-IR nerve fibers coming from the trigeminal ganglion appeared in the dental pulp and periodontal ligament. In the molar dental pulp, thick NPY-IR nerve fibers were observed within the nerve bundle, and some thin NPY-IR nerve fibers ran towards the odontoblast layer; very few NPY-IR nerve fibers were observed in the incisor pulp. In the periodontal ligament of molar, thick NPY-IR nerve fibers appeared at the alveolar part following combined CCI of IAN and SCGx. In the lingual portion of the periodontal ligament of the incisor, many thick NPY-IR nerve fibers were observed. These occasionally showed a tree-like appearance, resembling immature Ruffini endings; slowly adapting mechanoreceptors. The present results indicate that periodontal mechanoreceptors are among the main targets of injury-evoked NPY following IAN injury.

Growth-associated protein-43 (GAP-43) in the regenerating periodontal Ruffini endings of the rat incisor following injury to the inferior alveolar nerve

Alterations in the levels of growth-associated protein 43 (GAP-43)-like immunoreactivity (-LI) were examined in the lingual periodontal ligament of the rat incisor following two types of injury (resection and crush) to the inferior alveolar nerve (IAN). In normal animals, GAP-43-like immunoreactive (IR) structures were observed as tree-like ramifications in the alveolar half of the lingual periodontal ligament of incisors. Under immunoelectron microscopy, GAP-43-LI appeared in the Schwann sheaths associated with periodontal Ruffini endings; neither cell bodies of the terminal Schwann cells nor axonal profiles showed GAP-43-LI. During regeneration of the periodontal Ruffini endings following resection of the IAN, GAP-43-LI appeared in the cytoplasm of the terminal Schwann cell bodies and axoplasm of the terminals. The distribution of GAP-43-LI in the Ruffini endings returned to almost normal levels on days 28 and 56 following the injury. The changes in the distribution of GAP-43-LI following the crush injury were similar to those following resection; however, expression of GAP-43-LI was slightly higher for the entire experimental period compared with the resection. The transient expression of GAP-43 in the terminal Schwann cells and axonal profiles of the periodontal Ruffini endings following nerve injury suggests that GAP-43 is closely associated with axon-Schwann cells interactions during regeneration.

Calretinin-like immunoreactivity in the Ruffini endings, slowly adapting mechanoreceptors, of the periodontal ligament of the rat incisor

The distribution and ultrastructural localization of calretinin (CR)-like immunoreactivity (-LI) were investigated in the lingual periodontal ligament of rat incisors. Some thick nerve fibers within the nerve bundle displayed CR-LI; these CR-like immunoreactive (-IR) nerve fibers entered the alveolar half of the lingual periodontal ligament of the incisor where dendritic terminal arborization was exhibited. Thin and beaded CR-IR nerve fibers were rarely observed in the periodontal ligament. Observations of adjacent sections immunostained with protein gene-product 9.5 (PGP 9.5) revealed that most, if not all, PGP 9.5-IR nerve terminals showing a dendritic arborization expressed CR-LI. Immunoelectron microscopic observations showed that electron-opaque immunoreaction products were localized in the axoplasm of the axon terminals, except for the mitochondria, which were surrounded by Schwann sheaths and multiple-layered basal lamina. Neither cell bodies, the cytoplasmic extension of terminal Schwann cells, nor other cellular elements such as periodontal fibroblasts exhibited CR-LI. The present findings suggest that Ruffini endings, an essential mechanoreceptor in the periodontal ligament and categorized as a slowly adapting mechanoreceptor, express CR-LI, and that CR may participate in the Ca2+ homeostasis against external stimuli in the periodontal Ruffini endings.

Immunohistochemical localization of calbindin D28k in the periodontal Ruffini endings of rat incisors

It was examined whether calbindin D28k (CB) might be located in the rat incisor periodontal Ruffini ending, an essential mechanoreceptor in periodontal ligament, by light- and electron-microscopic immunohistochemistry. Some thick nerve fibers showing CB-like immunoreactivity (LI) entered the lingual half of the periodontal ligament of the incisor and showed the dendritic terminal arborization. Electron-dense immunoreaction products indicating CB-LI were distributed diffusely in axoplasm of the axon terminals, no mitochondria, however, were not labeled. Neither cell bodies nor cytoplasmic extensions of the terminal Schwann cells exhibited CB-LI. CB was presumed to be involved in the maintenance of Ca2+ homeostasis in the mechano-electric transduction in mechanoreceptors in the periodontal ligament.

Neuropeptide Y-like immunoreactive primary afferents in the periodontal tissues following dental injury in the rat

The distribution of neuropeptide Y (NPY)-like immunoreactive (-IR) nerve fibers in the periodontal tissues following dental injury to the rat maxillary first molar was examined with a combination of dental injury and surgical sympathectomy of the superior cervical ganglion (SCGx). In normal animals, NPY-IR nerve fibers were observed around the blood vessels of the trigeminal ganglion, dental pulp and periodontal tissues. These nerve fibers completely disappeared following SCGx. Fourteen days following dental injury of the maxillary first molar combined with SCGx, a small number NPY-IR cells was observed in the dorsal to middle portion of the maxillary division of the trigeminal ganglion. These were mostly medium- to large-sized cells with a mean +/- SD cross-sectional area of 541.4 +/- 239.3 microns 2. Approx. 50% of these cells had the cross-sectional areas between 400-600 micron 2. In the periodontal tissues of injured first molar, thick NPY-IR nerve fibers showing an irregular appearance were detected in the apical region. Immunoelectron microscopy showed that most NPY-IR nerve fibers near the lower half of the injured periodontal ligament had an axonal diameter of approx. 7-8 microns, and lacked apparent myelin sheaths. Near NPY-IR nerve fibers, many macrophages with phagosomes containing debris of the myelin sheaths were observed. At the oral epithelium covering the injured roots of the maxillary first molar, thick NPY-IR nerve fibers were recognizable and some penetrated the epithelium. No NPY-IR nerve fibers were observed in the dental pulp or periodontal tissues in second and third molars, and ultrastructural views of nerve fibers were almost intact following combined SCGx and dental injury to the first molar. The present results indicate that NPY-IR primary afferents appeared in the periodontal tissues following dental injury, and that NPY may be closely associated with the regeneration process of injured primary afferents.

Distribution and possible origin of galanin-like immunoreactive nerve fibers in the mammalian dental pulp

Human, dog, cat and rat dental pulps were investigated for the presence and distribution of galanin-like immunoreactive (-IR) nerve fibers, and the possible origin of pulpal galanin-IR nerve fibers in the rat was examined. Galanin-IR nerve fibers were present in the dental pulps of all species examined. Two types of galanin-IR nerve fibers were distinguished with regard to morphology; thin varicose nerve fibers and thick smooth-surfaced nerve fibers. Thin varicose galanin-IR nerve fibers were seen to run along the blood vessel in the human, dog and cat root pulp. In the coronal pulp, galanin-IR nerve fibers ran toward the odontoblastic layer but they did not form the subodontoblastic nerve plexus. In rat molar pulp, few galanin-IR nerve fibers were observed; the distribution of these nerve fibers was similar to those in human, dog and cat pulp. In contrast, many thick smooth-surfaced galanin-IR nerve fibers were observed near the blood vessels in incisor pulp of the rat; occasionally a few varicose galanin-IR nerve fibers were also observed. Transection of the inferior alveolar nerve or mandibular nerve caused complete disappearance of galanin-IR nerve fibers in rat dental pulp, while surgical sympathectomy of the superior cervical ganglion did not affect their distribution. The present results indicate that galanin-IR nerve fibers are present in the mammalian dental pulp, and that the intrapulpal galanin-IR nerve fibers in the rat originate from the trigeminal ganglion and are primary afferents.

Partial coexistence of neuropeptide Y and calbindin D28k in the trigeminal ganglion following peripheral axotomy of the inferior alveolar nerve in the rat

Immunohistochemistry was applied to examine the correlation between neuropeptide Y (NPY) and the two calcium binding proteins (CaBPs) parvalbumin (PV) and calbindin D28k (CB) in the trigeminal ganglion following peripheral axotomy of the inferior alveolar nerve (IAN) in the rat. Five days following transection and application of FluoroGold (FG) to the cut end of the IAN, approximately 14.8% (80/539) and 18.6% (90/483) of FG-labeled IAN neurons in the trigeminal ganglion showed PV-like immunoreactivity (-LI) and CB-LI, respectively. The mean +/- S.D. area of FG-labeled PV-like immunoreactive (-IR) cells (FG/PV-IR cells) and FG/CB-IR cells were 835.9 +/- 303.1 mu m2 and 712.7 +/- 246.0 mu m2, respectively. FG/PV-IR cells were significantly larger than FG/CB-IR cells. Fourteen days following peripheral axotomy of the IAN, NPY-LI appeared in the medium- to large-sized cells. Double immunostaining revealed that approximately 3.3% (52/1569) of NPY-IR cells in the axotomized trigeminal ganglion displayed PV-LI, while approximately 26.7% (371/1392) of NPY-IR cells displayed CB-LI. The mean +/- S.D. cross-sectional areas of PV-IR and CB-IR trigeminal ganglion cells displaying NPY-LI were 819.5 +/- 265.6 mu m2 and 766.5 +/- 279.7 mu m2, respectively. There were no significant differences in the cross-sectional areas either between NPY/PV-IR cells and NPY/CB-IR cells, or between FG/PV-IR cells and NPY/PV-IR cells, or between FG/CB-IR cells and NPY/CB-IR cells. The present results indicate that injury-evoked medium- to large-sized NPY neurons were a different population from large-sized PV neurons, and NPY was partly co-localized with CB.