Neural sodium voltage-gated channel beta subunit 3 and alpha subunit 7 distributions in mouse periodontal ligaments after experimental tooth movement

ABSTRACT

Purpose

Distributional changes of beta subunit 3 (SCN3B) and alpha subunit 7 (SCN7A) proteins of voltage-gated sodium channel proteins were investigated in the periodontal ligament (PDL) after experimental tooth movement to understand their roles in pain transmission and bone remodelling during orthodontic tooth movement.

Materials and methods

Immunohistochemical staining for SCN; SCN and tartrate-resistant acid phosphatase (TRAP); and SCN, calcitonin gene-related peptide (CGRP), and TRAP were performed on the PDL in the first molar teeth of mice at 1–14 days after experimental tooth movement.

Results

In intact animals, the PDL of the first molar tooth had only a few SCN3B-immunoreactive (−IR) nerve fibres. At 5 days, however, SCN3B-IR nerve fibres significantly increased in the compression area of the PDL. SCN3B-IR nerve fibres were occasionally detected in the vicinity of TRAP-positive multinuclear cells within the resorption lacunae. These nerve fibres appeared to contain CGRP-immunoreactivity. At 7–14 days after tooth movement, SCN3B-IR nerve fibres decreased in the compression area and were infrequent in both compression and tension areas. In contrast, SCN7A-IR nerve fibres decreased in the compression area after the experimental tooth movement.

Conclusions

Experimental tooth movement changed the distribution of SCNs in the PDL. SCN3B may be associated with nociceptive transmission and regulate activity of osteoclasts through release of CGRP from sensory nerve endings during orthodontic tooth movement.

KEYWORDS:

• Sodium voltage-gated channel
• experimental tooth movement
• immunohistochemistry

Introduction

Voltage-gated sodium channels (SCNs) open and close in response to membrane potential changes, controlling sodium ion diffusion for neuronal membrane depolarization [1,2]. These channels are transmembrane glycoprotein complexes composed of alpha and beta subunits [1,2]. In humans, the sodium voltage-gated channel beta subunit 3 (SCN3B) and alpha subunit 7 (SCN7A) genes encode voltage-gated sodium channel proteins [3–5]. Furthermore, subunit proteins are distributed throughout the central nervous system [6–8]. For example, SCN3B protein-containing neurons are scattered throughout the cerebral cortex and hippocampus [6], and SCN3B levels decrease in the hippocampus but not in the cerebral cortex of patients with temporal lobe epilepsy [6]. This reduction influences neuronal excitability in the hippocampus and is thought to cause epileptogenesis in the brain. Neurons in circumventricular organs express the SCN7A protein, which responds to an increase in extracellular sodium concentration in the cerebrospinal fluid [8]. In the periphery, SCNs are expressed in the dorsal root ganglion (DRG) [7,8]. SCN3B and SCN7A are located in sensory neurons with small and medium-to-large cell bodies, respectively [7,9]. Furthermore, SCNs have been associated with nociceptive transmission in the DRG. For instance, the SCN3B level increased in a chronic constriction injury model of neuropathic pain [10], and SCN7A expression increased in the DRG in animal models of cancer pain [7]. In addition, SCN7A is expressed in satellite and Schwann cells [11,12]. However, the presence and function of SCNs in sensory neurons and their peripheral tissues remain unclear.

The periodontal ligament (PDL) is innervated by sensory neurons whose cell bodies are localized to the trigeminal ganglion (TG). TG neurons send their peripheral axons to the PDL and terminate as free nerve endings [13], which are nociceptors that respond to chemical, thermal, and mechanical stimuli [13–16]. Orthodontic tooth movement causes the PDL to compress and stretch and induces alveolar bone remodelling [17]. Consequently, bone resorption and formation occur in the compression and tension areas, respectively. An increase in nerve fibres containing protein gene product 9.5 and calcitonin gene-related peptide (CGRP) has also been detected in the compression area [18,19]. Sprouting is associated with discomfort and pain during orthodontic tooth movement. Moreover, CGRP released from nerve terminals is thought to affect bone remodelling during orthodontic tooth movement [19], and in vitro studies have demonstrated that CGRP regulates osteoclast activity [9,20,21]. Furthermore, sodium channel activation induces CGRP release from nerve terminals [22].

We hypothesized that SCNs function in nociceptive transmission and bone remodelling during orthodontic tooth movement. Therefore, this study examined the effect of experimental tooth movement on SCN3B- and SCN7A-containing nerve fibres.

Materials and methods

Mouse model

We used 24 male C57BL/6 mice (7-week-old). The mice were housed in a controlled temperature (25 °C) environment with a 12-h light/dark cycle and were fed water and food (standard basal diet) ad libitum. The experimental tooth movement mechanical stimulation model was prepared by fixing a nickel-titanium wire of 0.012-inch diameter to the maxillary incisor using a composite resin as a dental filling. The right maxillary first molar was moved towards the palatal side for up to 14 days (Figure 1a) [23]. Mice without mechanical stimulation (i.e. intact animals, day 0) were used as controls.

Figure 1. Schematic drawings at 0 days (blue in a, b) and 14 days (red in a) after experimental tooth movement of the first molar tooth. A nickel-titanium wire with 0.012-inch diameter moves the first molar tooth to the palatal side (a). Panel b shows the cross section of the palatal root (R) in the first molar tooth with experimental tooth movement. The closed arrow indicates the direction of the force by the nickel-titanium wire. Buccal and palatal regions in the periodontal ligament (PDL) were analysed as tension (TA) and compression areas (CA), respectively. AB; alveolar bone.

Tissue preparation

PDL samples were obtained from four mice on day 0 and days 1, 3, 5, 7, and 14 after initiating the experimental tooth movement. The mice were anaesthetized with isoflurane and then transvascularly perfused with 10 mL of saline, followed by 100 mL of Zamboni fixative [24]. The maxillae of all mice were decalcified with 10% ethylenediaminetetraacetic acid (pH 7.4) for 1 week at room temperature. The maxilla was coronally sectioned in 8-µm slices and mounted on silane-coated glass slides for haematoxylin-eosin (HE) staining and immunohistochemistry assays.

Immunohistochemistry

The sections were incubated for 24 h at room temperature with rabbit anti-sera against SCN3B (1:4000, Atlas Antibodies AB, Stockholm, Sweden) and SCN7A (1:6000, Atlas Antibodies AB), followed by incubation for 2 h at room temperature with biotinylated goat anti-rabbit IgG and avidin-biotin-horseradish peroxidase complex (ABC stain; Vector Laboratories, Newark, CA). Immunoreaction products were visualized using diaminobenzidine and nickel ammonium sulphate. Tartrate-resistant acid phosphatase (TRAP) staining was performed on SCN3B-stained sections (Sigma Aldrich, St. Louis, MO). Other sections were incubated for 24 h at room temperature with rabbit anti-SCN3B serum (1:4000) for tyramide signal amplification (TSA; Akoya Biosciences, Marlborough, MA). After incubation with the TSA reagent, the cells were incubated for 24 h at room temperature with rabbit anti-CGRP serum (1:2000, Peninsula Laboratories, San Carlos, CA) and then for 2 h at room temperature with fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG (1:100, Jackson ImmunoResearch Laboratories, West Grove, PA). Subsequently, the sections were processed for TRAP staining [25].

The specificity of rabbit anti-CGRP serum has been described elsewhere [26]. An absorption test was conducted to determine the specificity of anti-SCN sera using SCN3B (100 mg/mL) and SCN7A (20 mg/mL) proteins. The control group had no immunoreactivity.

Morphometric analysis

The buccal and palatal sides of the PDL in the upper first molars were analysed (Figure 1b). Each histomorphometry group contained four mice, and two sections with 80, 160 and/or 240 μm distance from root furcation of the palatal root were randomly selected per animal to analyse the thickness of the PDL- and SCN-positive nerve fibres. The sections were situated in cervical 1/3 of the palatal root. So that, the direction of the press force by a nickel-titanium wire could be configured almost orthogonal to the tooth axis [23,27]. The lengths were divided by the length of the radicular surface in the compression and tension areas. The quotient was recorded for density. In addition, the total number of multinuclear cells was obtained from every six serial sections of the cervical half of the palatal root.

Statistical analyses

The differences between the compression and tension areas were analysed using t-tests.

Ethical approval

The experiments were performed under the control of our institution’s Animal Research Control Committee following the relevant university and national guidelines for the care and use of laboratory animals as well as the feeding and safekeeping of animals. Furthermore, all efforts were made to minimize the number of animals used and their suffering. All experiments were performed after being reviewed and approved by the Institutional Laboratory Animal Care and Use Committee of Tohoku University (2020DnA-013-03).

Results

PDL after experimental tooth movement

The PDL of intact first molars (i.e. day 0) had orderly arranged fibroblasts between the root cementum and alveolar bone, and resorption lacunae were infrequent (HE stain; Figures 1 and 2). However, on day 5 of experimental tooth movement, the PDL fibroblasts were compressed in the compression area and stretched in the tension area (Figures 2 and 3). Furthermore, the PDL thickness was significantly smaller in the compression area than in the tension area (Figure 3(a), *p < 0.05, t-test). On days 3 and 5, the compression area had several resorption lacunae, and large cells with multiple nuclei were usually detected in these areas (Figures 2 and 3, *p < 0.05, **p < 0.01, t-test). On days 7 and 14, the PDL thicknesses in the compression and tension areas were similar (Figures 2 and 3), and resorption lacunae and large cells were infrequently observed. At all stages of tooth movement, the tension area contained only a few resorption lacunae (0–2 cells). The surface of alveolar bone in tension side was smooth on the first day and became uneven thereafter, indicating that the bone formation appeared in tension side on days 3–14.

Figure 2. Microphotographs of haematoxylin-eosin-stained sections in the intact periodontal ligament (PDL) (a, b) and the PDL on days 1 (c, d), 3 (e, f), 5 (g, h), 7 (i, j), and 14 (k, l) after initiating tooth movement. After 5 days, compressed and stretched PDL fibroblasts are detectable in the compression (g) and tension (h) areas, respectively. Furthermore, some resorption lacunae (RL) are present in the compression area. After 14 days, no remarkable morphological changes to the PDL are observed. Arrowheads in g indicate large cells in the RL. R; root, AB; alveolar bone. Scale bar = 50 μm (a). All panels are at the same magnification.

Figure 3. Line graphs illustrating the periodontal ligament (PDL) thickness (a) and the number of large cells with multiple nuclei (b) in the intact PDL (day 0) and the PDL after 1, 3, 5, 7, and 14 days of tooth movement (four mice per stage).

SCN3B after experimental tooth movement

On day 0, the PDL of the first molar tooth had few SCN3B-immunoreactive (IR) nerve fibres, but the number of IR fibres significantly increased by day 5 (Figures 4 and 6). Furthermore, SCN3B-IR nerve fibres entered the resorption lacunae, ramified slightly, and migrated towards the surface of the alveolar bone. On days 7–14, the SCN3B-IR nerve fibres decreased in the compression area (Figures. 4 and 6). Changes in the tension area did not occur at any timepoint.

Figure 4. Microphotographs of sodium voltage-gated channel beta subunit 3 (SCN3B) in the intact periodontal ligament (PDL) (a, d) and the PDL after 1 (b, e), 3 (c, f), 5 (g, j), 7 (h, k), and 14 (i, l) days of the tooth movement. The intact PDL (day 0) has a few SCN3B-immunoreactive (IR) nerve fibres, whereas many nerve fibres are detected within the resorption lacuna (RL) in the compression area on day 5. The number of SCN3B-IR nerve fibres decreases 14 days after the tooth movement. R; root, AB; alveolar bone. Scale bar = 50 μm (a). All panels are at the same magnification.

Figure 5. Microphotographs of sodium voltage-gated channel alpha subunit 7 (SCN7A) in the intact periodontal ligament (PDL) (a, d) and the PDL after 1 (b, e), 3 (c, f), 5 (g, j), 7 (h, k), and 14 (i, l) days of tooth movement. The intact PDL (day 0) has many nerve fibres on the palatal side but few on the buccal side (a). After the tooth movement, SCN7A-immunoreactive (IR) nerve fibres decrease in the compression area (b, c, g-i). The distributions of SCN7A-IR nerve fibres in the compression and tension areas are similar on days 5 and 7 after tooth movement. R; root, AB; alveolar bone. Scale bar = 50 μm (a). All panels are at the same magnification.

Figure 6. Line graphs showing the distribution of sodium voltage-gated channel beta subunit 3-immunoreactive (SCN3B-IR) (a) and alpha subunit 7-immunoreactive (SCN7A-IR) (b) nerve fibres on days 0–14 of experimental tooth movement (four mice per stage).

After tooth movement, TRAP-positive cells were located close to the surface of the alveolar bone within resorption lacunae in the compression area. They typically had large cell bodies and multiple nuclei (from 2–5). Dual ABC and TRAP staining showed that fine SCN3B-IR nerve fibres ran towards TRAP-positive cells, occasionally terminating as free nerve endings near TRAP-positive cells (within 5 μm distance, Figures 7(a) and 8). SCN3B-IR nerve fibres in the vicinity of TRAP-positive cells were common on day 5, and relatively infrequent on other days. Many CGRP-IR nerve fibres are located in the PDL [13,28,29]. Thus, we performed a triple-stain analysis, finding that SCN3B-IR nerve fibres near TRAP-positive cells had CGRP immunoreactivity on days 3–7 (Figure 8).

Figure 7. Microphotograph of sodium voltage-gated channel beta subunit 3 (SCN3B) (a), alpha subunit 7 (SCN7A) (b) and tartrate-resistant acid phosphatase (TRAP) staining in the compression area of the PDL after 5 days of tooth movement. SCN3B-immunoreactive nerve fibres (arrow in a) are very close to a TRAP-positive cell (arrowhead in a). However, SCN7A-IR nerve fibres (arrow) were distant from a TRAP-positive cell (arrowhead in a). Scale bar = 20 μm.

Figure 8. Microphotographs of sodium voltage-gated channel beta subunit 3 (SCN3B) (a, c, d), and tartrate-resistant acid phosphatase (TRAP) (b, c), and calcitonin gene-related peptide (CGRP) (d) in the compression area of the PDL after 5 days of tooth movement. All panels are in the same field of view. Arrows in a-d show nerve varicosities containing SCN3B and CGRP. The varicosities are near a TRAP-positive multinuclear cell (arrowheads in b and c). Scale bar = 10 μm (a). All panels are at the same magnification.

SCN7A after experimental tooth movement

On day 0, the first molar tooth had thick and thin SCN7A-IR nerve fibres associated with nerve bundles and blood vessels (Figure 5). In addition, isolated nerve fibres with slight ramifications were also SCN7A-immunoreactive. These nerve fibres were numerous on the palatal side but infrequent on the buccal side of the PDL (Figures 5 and 6). However, experimental tooth movement decreased the amount of SCN7A-IR nerve fibres on the palatal side (i.e. the compression area). Dual ABC and TRAP staining showed that SCN7A-IR nerve fibres were always located more than 30 μm far from TRAP-positive cells (Figure 7(b)).

Discussion

This immunohistochemical study demonstrated that nerve fibres in the PDL of mice with and without experimental tooth movement contained SCN3B. In the TG, SCN3B-containing sensory neurons have mainly small cell bodies [9]. However, SCN3B in the autonomic ganglion has not yet been reported. Therefore, SCN3B-containing nerve fibres likely originate from small TG neurons.

Moreover, experimental tooth movement increased the number of SCN3B-containing nerve fibres. SCN3B is a subunit protein of voltage-gated SCNs that generates an action potential in sensory neurons; it is also involved in nociceptive transduction [10,30]. The expression of scn genes changes in the dorsal root ganglion of the transgenic mouse that constitutively expresses tumour necrosis factor (TNF) [30]. The mouse is an established model of chronic systemic inflammation, and shows increased sensitivity to mechanical and thermal heat stimulation. And the hypersensitivity is thought to be associated with upregulation of scn3b gene in small dorsal root ganglion neurons. Compared to the tension side, the orthodontic tooth movement increases expression of TNF-alpha but not TNF-beta in the compression side [31]. It is possible that upregulation of TNF-alpha induces sprout of SCN3B-IR nerve fibres and change of sensitivity to the mechanical force. During orthodontic tooth movement, patients experience pain or discomfort due to continuous pressure on the PDL [32,33]. Therefore, more SCN3B-containing nerve fibres by TNF-alpha may reflect an uncomfortable feeling during orthodontic tooth movement.

Furthermore, SCN3B-containing nerve fibres increased in the compression area but not in the tension area. These nerve fibres were near TRAP-positive cells with large cell bodies and multiple nuclei in the resorption lacunae, and our triple-stain analysis demonstrated that they co-expressed with CGRP. TRAP-positive cells are likely osteoclasts that absorb alveolar bone in the compression area, and CGRP enhances osteoclast activity during tooth movement [34]. Therefore, the co-expression of SCN3B and CGRP suggests that SCN3B may regulate osteoclast activity through CGRP release from sensory nerve endings. Previous studies reported that experimental tooth luxation and movement increases CGRP-containing nerve fibres near osteoclasts with receptor activity modifying protein 1 (RAMP1), a CGRP receptor subunit [13,28], supporting our theory.

Intact animals (i.e. day 0) had more SCN7A-containing nerve fibres on the palatal side than on the buccal side of the PDL. Then, PDL compression during tooth movement decreased the neural SCN7A protein content. Compared to the tension side, matrix metalloproteinase-1 (MMP-1) expression is increased in the compression side of the human PDL during tooth movement [31]. Upregulation of MMP-1 can induce cytotoxicity in human Schwann cells [35]. Therefore, decrease of SCN7A-IR nerve fibres in the compression side during tooth movement may be due to degeneration or death of SCN7A-IR Schwann cells induce by MMP-1 upregulation.

By previous and present findings, the tooth movement causes osteoblast activation and new bone formation in the tension area [25]. In the tension area, the orthodontic tooth movement induces increase of some cytokines such interleukin-10 and tissue inhibitor of metalloproteinase-1 [31]. Nevertheless, no significant change of the density of SCN3B- and SCN7A-IR nerve fibres could not be detected in the tension side of the PDL during the tooth movement. The PDL expansion, new bone formation and cytokine upregulation in the tension area may have little or no effect on contents of SCN3B- or SCN-7A proteins, sprouting of peripheral neurites and survival of Schwann cells.

Bone tissue remodelling processes are regulated by various cytokines such as TNF-alpha and MMP-1 [36–38]. Theses cytokines may control bone remodelling as well as nerve sprouting or degeneration in the PDL during tooth movement. However, the functional significance of SCN7A and in the PDL remains still unclear. Further studies are necessary to determine the origin and function of SCN3B and SCN7A in PDL with and without tooth movement.

Conclusion

Experimental tooth movement increased the amount of SCN3B-IR nerve fibres and decreased the amount of SCN7A-IR nerve fibres in the PDL of mice. Furthermore, SCN3B-IR nerve fibres co-expressing CGRP were present near TRAP-positive multinuclear cells within the resorption lacunae in the compression area. SCN3B may be associated with nociceptive transmission and regulate osteoclast activity via CGRP release from sensory nerve endings during orthodontic tooth movement.

Author contributions

Author 1 and 2 designed the study, conducted acquisition of data and data analysis, completed the final version of the manuscript.

Author 3, 4, 5, 6 and 8 conducted acquisition of data and data analysis, completed the final version of the manuscript.

Author 7 and 9 designed the study, conducted acquisition of data and data analysis, wrote the draft version of the manuscript, completed the final version of the manuscript.

Ethical approval

The experiments were performed under the control of our institution’s Animal Research Control Committee following the relevant university and national guidelines for the care and use of laboratory animals as well as the feeding and safekeeping of animals. All experiments were performed after being reviewed and approved by Institutional Laboratory Animal Care and Use Committee of Tohoku University (2020DnA-013-03).

Acknowledgments

We thank all lab members for their helpful assistance and comments. We would like to thank Editage (www.editage.com) for English language editing.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by JSPS KAKENHI [grant numbers 21K10154 and 22K17243].