Increase of CGRP-Containing Nerve Fibers in the Rat Periodontal Ligament After Luxation

Abstract

The distribution of calcitonin gene-related peptide (CGRP) was examined in the periodontal ligament (PDL) after experimental luxation injury of the rat first molar tooth. The luxational injury increased the number of CGRP-immunoreactive (IR) nerve fibers. At 3–7 days, numerous CGRP-IR nerve fibers appeared throughout the injured PDL. These nerve fibers terminated as free nerve endings within resorption cavities. Immunohistochemistry for receptor activity modifying protein 1 (RAMP1) also demonstrated that the subunit of CGRP receptor was expressed by periodontal cells adjacent to the alveolar bone in the intact and injured PDL. RAMP1-IR cells were divided into two types; small cells with single nucleus and large cells with 2–6 nuclei. After the luxational injury, both types of RAMP1-IR cells abundantly appeared within resorption cavities. As a result, the treatment increased the number of large RAMP1-IR cells at 3–7 days and small RAMP1-IR cells at 7 days. In addition, a double immunofluorescence analysis demonstrated that CGRP-IR nerve fibers were seen away from RAMP1-IR cells in the intact PDL. After the traumatic injury, however, CGRP-IR nerve fibers appeared in the close vicinity of small and large RAMP1-IR cells at 5–7 days. The morphology and distribution of RAMP1-IR cells suggest that they contain osteoblasts and osteoclasts. By affecting osteoclasts and osteoblasts, CGRP may have effects on bone remodeling in the luxated PDL.

Introduction

Calcitonin gene-related peptide (CGRP) is a neuropeptide which is widely distributed in the central and peripheral nervous systems (Skofitsch and Jacobowitz 1985; Silverman and Kruger 1989; Sugimoto et al. 1997). In the trigeminal ganglion (TG), CGRP-containing primary sensory neurons have small to medium-sized cell bodies (Ichikawa and Sugimoto 2003). They project to superficial laminae of the medullary dorsal horn (Sugimoto et al. 1997). In the periodontal ligament (PDL), CGRP-containing TG neurons send their peripheral axons to blood vessels and terminate as free nerve endings (Kvinnsland and Kvinnsland 1990; Norevall et al. 1995). CGRP is considered to be associated with transmission of nociceptive information from the PDL. Previous immunohistochemical studies have demonstrated the effect of experimental tooth movement on CGRP-containing nerve fibers (Kvinnsland and Kvinnsland 1990; Norevall et al. 1995). At 5–7 days after the treatment, the number of CGRP-containing nerve fibers increases in the PDL. In vitro experiments, CGRP has been shown to regulate the activity of osteoblasts and osteoclasts (Naot and Cornish 2008; Wang et al. 2010). Therefore, this peptide is also considered to be involved in bone remodeling during experimental tooth movement.

The luxation of the human tooth causes tissue damages and reactions in the PDL. To understand the changes, experimental models in which traumatic injuries are given to dental tissues have been developed (Miyashin et al. 1990, 1991). The rat molar tooth is luxated by tooth separation with a dental forceps or partial tooth extraction with a dental elevator. After the trauma, irregular periodontal fibers, degenerated cells, hemorrhage, and edema are detected in the PDL (Miyashin et al. 1990, 1991). In addition, the alveolar bone resorption severely occurs from 1 day after the injury. Then, the bone gradually repairs. Thus, it is likely that the luxational trauma results in active remodeling of the alveolar bone. However, the distribution of CGRP in the PDL has not been reported after tooth luxation.

In the present study, we examined CGRP-immunoreactive (IR) nerve fibers in the PDL of rat luxated teeth. The distribution of CGRP receptor was also investigated by immunohisochemistry for its subunit, receptor activity modifying protein 1 (RAMP1) (McLatchie et al. 1998; Miyauchi et al. 2002). In addition, a double immunofluorescence method was performed to know the relationship between CGRP-IR nerve fibers and RAMP1-IR cells in the PDL.

Experimental Procedure

Forty-eight adult male Sprague-Dawley rats (180 g) were used in this study. For partial extraction of the left upper first molar tooth, its distal edge was raised at 0.5 mm distance using an original elevator with a curved tip (thickness = 1 mm, length = 3 mm) in 40 animals under deep anesthesia by i.p. injection with pentobarbital sodium (40 mg/kg). Intact and operated animals after 1, 3, 5, 7, and 14 days were reanesthetized with ether, and transvascularly perfused with Zamboni fixative. The maxillae were dissected and post-fixed with the same fixative for 30 min. Then, these materials were decalcified with 4.13% ethylene diaminetetraacetic acid disodium salt in 0.1 M phosphate buffer (pH 7.4) for 3 weeks at room temperature, and soaked in a phosphate-buffered 20% sucrose solution overnight. For CGRP-immunohistochemistry, the materials were frozen sectioned at 40 μm, and treated as free-floating sections. For hematoxylin-eosin (HE) stain or RAMP1 immunohistochemistry, they were cut at 8 μm with a cryostat, and thaw-mounted on gelatin-coated glass slides.

For demonstration of CGRP or RAMP1, ABC (avidin–biotin–horseradish peroxidase complex) method was performed. Sections were incubated with rabbit anti-CGRP (1:100000, Pennisula Lab., USA) or RAMP1 (1:750, Santa Cruz Biotechnology Inc., USA) sera, followed by biotinylated goat anti-rabbit IgG and ABC (Vector Laboratories, USA). Following nickel ammonium sulfate-intensified diaminobenzidine reaction, the sections were dehydrated in a graded series of alcohols, cleared in xylene and cover-slipped with Entellan (Merck, Germany). An indirect immunofluorescence method was also used for detection of RAMP1. Sections were incubated with rabbit anti-RAMP1 serum (1:25) and lissamine rhodamine B chloride-conjugated donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch Labs, USA). For simultaneous visualization of CGRP and RAMP1 in the PDL, a double immunofluorescence method was used. The sections were incubated for 24 h at room temperature with a mixture of rabbit anti-RAMP1 serum (1:25) and guinea-pig anti-CGRP serum (1:500, Milab, Sweden). The sections were then treated with a mixture of lissamine rhodamine B chloride-conjugated donkey anti-rabbit IgG (1:500) and DyLight488-conjugated donkey anti-guinea-pig IgG (1:100; Jackson ImmunoResearch Labs). Immunofluorescent sections were viewed with a fluorescent microscope or a confocal laser scanning microscope (Nikon, Japan) at the slice thickness of 0.5 μm.

For quantitative morphometric analysis of periodontal CGRP-IR nerve fibers, ABC-stained images were stored into a personal computer and the length of positive nerve fibers was measured in every third section of contiguous sections of the first molar tooth with NIS ELEMENTS software (Nikon, Japan). For quantitative morphometric analysis of periodontal RAMP1-IR cells, the number of positive cells was counted in every tenth section of contiguous sections of the first molar tooth. All the difference was analyzed with ANOVA and Bonferroni-type multiple t test.

The specificities of antibodies used in this study have been described elsewhere (Ichikawa et al. 1990; Nikitenko et al. 2001). In addition, normal rabbit serum was used instead of the primary antibodies. No immunostaining was detected for the control.

The experiments were carried out under the control of the Animal Research Control Committee in accordance with the guidelines for Animal Experiments of Tohoku University, Government Animal Protection and Management Law (No. 105), and Japanese Government Notification on Feeding and Safekeeping of Animals (No. 6). All efforts were made to minimize the number of animals used and their suffering.

Results

Hematoxylin–Eosin Stain

In the intact first molar, periodontal fibroblasts arranged orderly between the root cementum and the alveolar bone (Fig. 1a). Small resorption cavities were detected on the distal but not the mesial surface of the alveolar bone. Blood vessels and nerve bundles were also distributed in the intact PDL (Fig. 1a). Osteoclasts were occasionally located within resorption cavities, whereas osteoblasts were abundantly observed adjacent to the alveolar bone.

Fig. 1.

Microphotographs of hematoxylin and eosin stain in the PDL of intact (a) and luxated (b–f) animals. In the intact tooth (a), a small resorption cavity (CA, an arrow) is detected on the alveolar bone surface of the distal PDL. At 3 (b) and 7 (d) days after the luxation, numerous resorption cavities with various sizes (CA, arrows) appeared throughout the PDL. Areas of squares in Panels b and d represent microscopic fields of view in inset c and e, respectively. Arrows in inset c and e indicate osteoclasts and osteoblasts on the bone surface, respectively. At 14 days, the resorption cavities are rare and the alveolar bone surface becomes smooth (f). AB = alveolar bone. C = cementum. Bars 100 μm (a, b, d, f), 20 μm (c) and 10 μm (e)

At 1 and 3 days after partial extraction of the first molar tooth, fibroblasts in the injured PDL showed an irregular arrangement. The PDL contained hemorrhage at 1–5 days and inflammation at 1–7 days after the treatment. At 3–7 days, numerous resorption cavities with various sizes appeared throughout the alveolar bone surface (Figs. 1b, d). Small blood vessels and nerve bundles were located within these cavities. In addition, the cavities commonly contained osteoclasts at 3–5 days and osteoblasts at 5–7 days after the injury (Figs. 1c, e). At 14 days, however, the resorption cavities mostly disappeared and the alveolar bone surface became smooth (Fig. 1f).

CGRP-Immunohistochemistry

The PDL of the first molar tooth in intact animals had CGRP-IR nerve fibers (Fig. 2a). These nerve fibers were seen within thick and thin nerve bundles. Some CGRP-IR nerve fibers separated from the nerve bundles, ran toward blood vessels and surrounded them. In addition, CGRP-IR nerve fibers occasionally terminated as free nerve endings. The perivascular nerve fibers and free nerve endings were located within alveolar half of the PDL (Fig. 2b). However, CGRP-IR nerve fibers were very rare adjacent to the alveolar bone surface. Mean ± S.E. of the length of CGRP-IR nerve fibers in the intact PDL was 87.0 ± 20.9 mm (range = 33.2–126.5 mm) (Fig. 3).

Fig. 2.

Microphotographs of CGRP in the PDL of intact (a, b) and luxated (c, d) animals. In the intact tooth, CGRP-IR nerve fibers are distributed in the alveolar half of the PDL (arrows in a). However, these nerve fibers are very rare within the resorption cavity (CA in b). At 7 days after the luxation, the number of CGRP-IR nerve fibers increase throughout the PDL (arrows in c). They are abundant in the vicinity of the alveolar bone surface within resorption cavities (CA in d). AB = alveolar bone. C = cementum. Bars = 100 μm (a, b, d, F) and 20 μm (c, E)

Fig. 3.

Line graphs for total length of CGRP-IR nerve fibers in the PDL of intact (0 day) and luxated (1–14 days) animals. The number of CGRP-IR nerve fibers significantly increases at 5 and 7 days after the luxation compared with intact animals (*p < 0.05, ANOVA and Bonferroni-type multiple t test). The data were obtained from 4 intact and 20 luxated animals

The partial extraction of first molar teeth increased the number of CGRP-IR nerve fibers in the PDL. At 3–7 days after the treatment, numerous CGRP-IR nerve fibers appeared throughout the injured PDL (Fig. 2c). They branched repeatedly, ran toward the alveolar bone and terminated as free nerve endings within resorption cavities (Fig. 2d). As a result, the length of CGRP-IR nerve fibers in the PDL significantly increased at 5 days [mean ± S.E. = 180.7 ± 23.9 mm (108% increase), range = 132.8–226.4 mm] and 7 days [266.0 ± 42.9 mm (206% increase), 175.1–380.3 mm] after the injury (Fig. 3). At 14 days, however, CGRP-IR nerve fibers in the vicinity of the alveolar bone decreased and mean ± S.E. of the length of CGRP-IR nerve fibers in the inured PDL was 161.2 ± 12.4 mm (range = 140.4–197.3 mm) (Fig. 3).

RAMP1-Immunohistochemistry

RAMP1-IR cells were observed in the PDL of first molar teeth of intact and operated animals. These cells were located close to the alveolar bone. On the morphological basis, they were divided into two types; small and large types (Fig. 4a, c). The diameter of small RAMP1-IR cells ranged from 7.9 to 12.2 μm (mean ± S.D. = 9.7 ± 2.2 μm, n = 60). This type of RAMP1-IR cells had round or oval cell bodies with single nucleus. The diameter of large RAMP1-IR cells measured from18.8 to 33.1 μm (mean ± S.D. = 26.8 ± 3.6 μm, n = 30). Cell bodies of this type had irregular appearances, and contained 2–6 nuclei. The number of small RAMP1-IR cells was more numerous than that of large RAMP1-IR cells in intact and operated animals.

Fig. 4.

Microphotographs of RAMP1-IR cells (a, c) and line graphs for total number of RAMP1-IR cells (b, c) in the intact (0 day) and luxated (1–14 days) animals. Adjacent to the alveolar bone (AB in a, c) surface, RAMP-IR cells are divided into two types; large multi-nuclear types (arrows in a) and small mono-nuclear types (arrows in c). The luxation increases the number of large multi-nuclear RAMP1-IR cells at 3–7 days (a, b) and small mono-nuclear RAMP1-IR cells at 7 days (c, d) compared with intact animals (*P < 0.05, ANOVA and Bonferroni-type multiple t test). The data were obtained from 4 intact and 20 luxated animals

In the intact PDL, small RAMP1-IR cells were scattered in the vicinity of the alveolar bone surface, whereas large RAMP1-IR cells were located within small resorption cavities. Mean numbers ± S.E. of small and large RAMP1-IR cells were 4,891 ± 1,159 (range = 1,446–6,480) and 285 ± 59 (range = 129–380), respectively (Fig. 4b, d).

The partial extraction of first molar teeth affected the number of RAMP1-IR cells. At 3–7 days after the treatment, numerous large RAMP1-IR cells appeared within resorption cavities (Figs. 4a, b). As a result, their number was dramatically elevated in the injured PDL compared with the intact PDL [3 days, mean ± S.E. = 1,217 ± 137 (327% increase), range = 943–1,483; 5 days, mean ± S.E. = 1,573 ± 173 (453% increase), range = 1,187–1,939; 7 days, mean ± S.E. = 1,005 ± 69 (253% increase), range = 842–1,174] (Fig. 4b). At 7 days, the treatment also increased the number of small RAMP1-IR cells within resorption cavities [mean ± S.E. = 10,453 ± 804 (114% increase), range = 9,484–12,861] (Fig. 4d). At 14 days, both types of RAMP1-IR cells decreased; mean numbers ± S.E. of small and large RAMP1-IR cells were 4,809 ± 783 (range = 3,461–6,910) and 538 ± 51 (range = 435–626), respectively (Figs. 4b, d).

RAMP1-IR was also detected in the wall of large blood vessels. However, small blood vessels were devoid of RAMP1-IR.

Double Immunohistochemistry

A double immunofluorescence analysis demonstrated the relationship between CGRP-IR nerve fibers and RAMP1-IR cells in the PDL. Like in intact animals, such nerve fibers are seen away from RAMP1-IR cells within 3 days after the partial extraction. The distance between these profiles always measured > 20 μm. At 5 and 7 days after the treatment, however, CGRP-IR nerve fibers appeared in the close vicinity of small and large RAMP1-IR cells (Figs. 5a, b). These nerve fibers were usually associated with small mono-nuclear RAMP1-IR cells. CGRP-IR nerve fibers adjacent to large multi-nuclear RAMP1-IR cells were relatively rare in the injured PDL.

Fig. 5.

Microphotographs of CGRP and RAMP1 in double stained sections of luxated animals by a fluorescent microscopy (a) and a laser scanning microscopy (b). CGRP-IR nerve fibers (arrowheads) are located in the vicinity of large multi-nuclear RAMP1-IR cells (arrows in a) and small mono-nuclear cells (arrows in b). Bars 50 μm

Discussion

As described previously (Kvinnsland and Kvinnsland 1990; Norevall et al. 1995), CGRP-immunoreactivity was detected in perivascular nerve fibers and free nerve endings within the PDL of the first molar tooth in intact animals. After partial extraction of the tooth, many CGRP-IR nerve fibers appeared in the PDL. These nerve fibers terminated in the vicinity of the alveolar bone surface. The distribution and morphology of the CGRP-IR nerve fibers in the injured PDL suggest that they at least partly have a sensory nature and originate from the sensory ganglion, i.e., the trigeminal ganglion. CGRP may be associated with nociceptive transmission in the injured PDL. In this study, the luxational trauma induced inflammation in the PDL at 1–7 days. In previous studies, CGRP has been shown to be a vasodilator and play an important role in neurogenic inflammation (Gamse et al. 1987; Baraniuk et al. 1990; Györfi et al. 1992). Thus, this peptide may also have an effect on local blood flow and the inflammatory response in the injured PDL.

The functional receptor for CGRP is a complex form of RAMP1 and calcitonin receptor-like receptor (Naot and Cornish 2008). Previous studies have demonstrated that these proteins are expressed by osteoblasts in the human as well as bone marrow cells in the human and mouse (Uzan et al. 2004; Granholm et al. 2008). Osteoclast-like cells from co-cultures of mouse bone marrow cells and spleen cells are devoid of RAMP1 (Granholm et al. 2008). However, CGRP is known to have effects on rat osteoclasts (Naot and Cornish 2008; Wang et al. 2010). Thus, there appears to be species difference about osteoclastic RAMP1 expression. The present study demonstrated that RAMP1 is expressed by periodontal cells adjacent to the alveolar bone in intact and injured teeth. The morphology and distribution of small mono-nuclear and large multi-nuclear RAMP1-IR cells were similar to those of osteoblasts and osteoclasts, respectively. Therefore, periodontal RAMP1-IR cells probably contain osteoblasts and osteoclasts. The experimental luxation injury may increase the number of RAMP1-IR osteoblasts and osteoclast in the PDL.

Many CGRP-IR nerve fibers appeared adjacent to the alveolar bone at 5–7 days after the luxational trauma. The injury also increased the number of large multi-nuclear RAMP1-IR cells at 3–7 days and small mono-nuclear RAMP1-IR cells at 7 days. Double immunofluorescence analysis demonstrated that CGRP-IR nerve fibers were located adjacent to the RAMP1-IR cells at 5–7 days after the injury. These findings suggest that CGRP has functions to RAMP1-IR cells after experimental luxation injury. Previous studies have indicated that CGRP inhibits osteoclast activity and bone resorption (Naot and Cornish 2008; Wang et al. 2010). This peptide is also known to stimulate osteoblast proliferation in vitro and increase bone formation in vivo (Wang et al. 2010). By affecting osteoclasts and osteoblasts, CGRP may suppress bone resorption and promote bone formation after the experimental luxation injury. In addition, CGRP-IR nerve fibers were usually associated with small RAMP1-IR cells (osteoblasts), whereas CGRP-IR nerve fibers in the vicinity of large RAMP1-IR cells (osteoclasts) were relatively rare. These findings may suggest that CGRP has more important function to bone formation compared with bone resorption after the tooth luxation.

Amylin (islet amyloid polypeptide) is a 37 amino acid polypeptide which bears 47% structural homology with CGRP (Westermark et al. 1986; Cooper et al. 1987). Amylin and CGRP are co-expressed by primary sensory neurons and their peripheral axons (Mulder et al. 1995). In addition, these peptides have been considered to have similar functions to bone remodeling (Hoff et al. 2002). Like CGRP, amylin can increase osteoblast proliferation and decrease osteoclast activity. Recently, calcitonin receptor has been demonstrated to form heterodimetric complexes with RAMPs (McLatchie et al. 1998; Miyauchi et al. 2002; Nakamura et al. 2005). Heterodimers of calcitonin receptor with RAMP1, RAMP2, and RAMP3 exhibit the pharmacological profiles of receptors specific for amylin (Roh et al. 2004). In the PDL, the presence of amylin or its receptor has not been reported. However, it is possible that amylin receptor was also detected by the present RAMP1 immunohistochemistry. Further studies will be necessary to know the distribution and function of amylin in the intact and injured PDL.