Abstract
Objective
Previous studies of nerve distribution in the orofacial complex have focused primarily on the anatomic courses of nerve fibers and have rarely addressed the density of nerve distribution. The nerve distribution in the mandible was described in only one report which showed an increase in nerve distribution density moving from the alveolar crest toward the inferior alveolar nerve. However, no previous reports have focused on the nerve distribution density in the maxilla.
Methods
In this study involving 6 Wistar rats, tissue samples from the maxillary molar region were obtained and sectioned for staining and analysis. All nerves including nociceptive nerves in 5 regions of the maxilla samples, ranging from the alveolar crest to the superior alveolar nerve, were immunohistochemically stained with antibodies to protein gene product (PGP) and calcitonin gene-related peptide (CGRP) to identify and measure the vertical and horizontal nerve distribution densities in the 5 regions under an optical microscope. We also determined the ratio of nociceptive (CGRP-positive) nerves to all (PGP-positive) nerves.
Results
The densities of both PGP- and CGRP-positive nerves increased vertically from the alveolar crest to the superior alveolar nerve and horizontally from the periosteal side to the periodontal ligament side within the maxilla. The ratio of CGRP- to PGP-positive nerves was on average greater than 80%.
Conclusion
These findings were consistent with those of the previous report on mandibular nerve distribution. However, our results suggest that the overall nerve distribution density in the maxilla is approximately one-third of that in the mandible. The results of this study, which report the nerve distribution density in the posterior maxilla, may support future research on pain and local anesthesia.
Keywords: Nociceptive nerve, Maxilla, PGP9.5, CGRP, Nerve distribution, Rat
Previous studies on the maxillary and mandibular nerves have focused mostly on the effects of local anesthetics and the anatomic pathways of nerves in humans.1–4 Kudo et al5 statistically analyzed nerve distribution densities in different regions of rat mandibles and reported that the nerve distribution density increased significantly moving from the alveolar crest toward the inferior alveolar nerve, which is situated deeper. Also in the mandible, the ratio of nociceptive nerves to all nerves was greater than 70%, with fewer voluntary motor nerves and more sensory afferent nerves. However, no previous studies have focused on the nerve distribution density in the maxilla of rats.
If the maxilla is innervated similarly to the mandible, we hypothesized that the nerve distribution density will increase vertically toward deeper, thicker nerves and horizontally toward the periodontal ligament. Furthermore, similar to the mandible, we speculated that the maxilla would have a smaller proportion of motor nerves and a larger proportion of nociceptive nerves. However, these predictions are not certain.
In this study, the maxillary peripheral nerves of rats were stained with specific protein gene product (PGP) for immunohistochemical analysis. Furthermore, to investigate the innervation of rat maxillas, we used calcitonin gene-related peptide (CGRP) specific to myelinated Aδ fibers and unmyelinated C fibers, which are believed to be associated with transmission of pain signals. Moreover, we measured the distribution density of all nerves, including Aδ and C fibers, and statistically compared the densities in 5 maxillary regions. The primary objective of this study was to examine changes in nerve distribution density in the vertical and horizontal dimensions in the molar region of rat maxillas. The secondary objective was to examine the ratio of nociceptive nerves (CGRP-positive) to all nerves (PGP-positive) within the molar region.
METHODS
All experiments were conducted according to the Animal Experiment Regulations of Ohu University6 and approved by the Ohu University Animal Experiment Committee concerning the ethics of animal experiments (Animal experiment permit number 2020-9, 2021-3). The authors have no conflicts of interest to declare.
Six male Wistar rats (10 weeks old; 270 ± 10 g) were used in the present study (Charles River, Inc). The rats were housed at room temperature (23°C) and 60% humidity with free access to food (MF, Oriental Yeast, Inc) and water until the day of experimentation.
Sample Preparation and Immunohistochemical Staining
The rats were anesthetized with an intraperitoneal injection of thiopental sodium (15 µg/g), thoracotomized, and perfused with saline through the left ventricle. The molar part of the right half of the maxillary bone was excised following perfusion fixation with phosphate buffer 4% paraformaldehyde (pH 6.2). The excised maxilla was immersed overnight in the same fixative at 4°C and decalcified with 10% EDTA solution (pH 7.0). The specimens were decalcified with 10% EDTA solution (pH 7.0) for 1 to 2 months. The decalcified specimens were embedded in paraffin, and 20 µm sections were obtained from the first maxillary molar region using a microtome. The specimens consisted of 24 uninterrupted, intact samples that were parallel to the sagittal plane and extended from the alveolar crest to the superior alveolar nerve.
The paraffin-embedded sections were deparaffinized and treated with 0.3% H2O2-containing methanol for 15 minutes to inactivate the endogenous peroxidase, blocked with normal goat or rabbit serum (VECTASTAIN Elite ABC kit, Vector Lab, Inc) for 60 minutes, and then reacted with a primary antibody. Rabbit anti-human protein gene product (PGP) 9.5 antibody (1:1000 dilution; 14730-1-AP, Proteintech, Inc) was used as the primary antibody for all nerves.7 Goat anti-rat calcitonin gene-related peptide (CGRP) antibody (1:500 dilution; LS-C41796, LSBio, Inc) was used as the primary antibody for nociceptive nerves.8
The sections were reacted with the primary antibodies at room temperature for 14 hours. The sections were then reacted with a secondary antibody, a biotin-labeled goat anti-rabbit antibody (VECTASTAIN Elite ABC kit, Vector Lab, Inc), at room temperature for 60 minutes, followed by a reaction with peroxidase-conjugated streptavidin (VECTASTAIN Elite ABC kit, Vector Lab, Inc) for 60 minutes. Immunoreactivity was visualized with a 0.3% H2O2 solution containing 0.05% 3.3-diaminobenzidinetet hydrochloride (DAB; Peroxidase Substrate Kit, Vector Lab, Inc). The nuclei were stained using 5% methyl green (Muto Pure Chemicals Co, Inc). For washing and dilution of antisera, a 0.05-M Tris-HCI buffer (pH7.6) was used.
In the negative control reaction, normal goat or rabbit serum (VECTASTAIN Elite ABC kit, Vector Lab, Inc) was used instead of primary antibody.
All sections were subsequently observed using light microscopy, and all images were captured on a personal computer. The interpretations of PGP-positive and CGRP-positive nerves by classification of nerves9 were shown in Table 1, and our findings were classified based on the referenced chart.
Table 1.
Interpretations of PGP- and CGRP-Positive Nerves
| Nerve classification | Fiber type | PGP | CGRP |
|---|---|---|---|
| Somatic | |||
| Motor (efferent) | Aα | + | − |
| Aγ | + | − | |
| Sensory (afferent) | Aα | + | − |
| Aβ | + | − | |
| Aδ (fast pain) | + | + | |
| C (slow pain) | + | + | |
| Autonomic | |||
| Sympathetic | B | + | − |
| Parasympathetic | C (visceral pain) | + | + |
PGP, protein gene product; CGRP, calcitonin gene-related peptide; PGP-positive nerves, all nerves; CGRP-positive nerves, nociceptive nerves (i.e., Aδ and C fibers). The classifications and definitions in this table are from references.7–9
Distribution Density Measurement of the Maxillary Nerves
For analysis, the maxillary bone of each sample was divided into 5 main regions, A to E (Figure 1). Moreover, the C region was subdivided horizontally into equal thirds (Figure 1). The periodontal ligament one-third was named C-Ligament side (CL), the center one-third was named C-Center (CC), and the periosteal one-third was named C-Periosteal side (CP). The area of the maxillary bone was measured and calculated for each region (A, B, C, D, E, CL, CC, and CP), and the number of nerves (PGP-positive and CGRP-positive) in each region was measured with image analysis software ZEN blue (Carl Zeiss) (Figure 2). The nerve distribution density was determined based on the number of nerves per area of bone (No. of nerves/mm2).
Figure 1.
Cross-sectional Specimen of Rat Maxillary Molar Alveolar Bone
The enclosed region is the alveolar bone which was divided vertically into 5 sections (A, B, C, D, and E) spanning from the alveolar crest to near the superior alveolar nerve. Region C was subdivided horizontally into equal thirds (CL, CC, and CP) spanning the periosteal side to periodontal ligament side. Nerve distribution density was measured in each region (No. of nerve fibers/mm2).
Figure 2.
Immunohistochemical Staining With Antibodies
The black arrows indicate nerves. The left image shows a specimen stained with protein gene product (PGP) antibody, while the right image shows a specimen stained with calcitonin gene-related peptide (CGRP) antibody. The area of bone in each region was measured, and the number of nerves per area was measured using ZEN blue image analysis software.
Statistical Analysis
The mean distribution density of PGP- and CGRP-positive nerves was statistically compared in 5 regions (A, B, C, D, and E) and 3 subregions (CL, CC, and CP). Additionally, the ratio of CGRP- to PGP-positive nerves was compared in each region. A χ2 test was used for statistical analysis, and a P < .05 was considered statistically significant.
RESULTS
Analysis of the vertical nerve distribution data showed that the mean nerve distribution density of PGP-positive and CGRP-positive nerves significantly increased vertically from region A to region E, moving from the alveolar crest upward toward the superior alveolar nerve (P < .001; Table 2; Figure 3).
Table 2.
Nerve Distribution Density and CGRP/PGP Ratio in Each Region
|
Vertical direction
| ||||||
|---|---|---|---|---|---|---|
|
Regions
|
P value | |||||
| A | B | C | D | E | ||
| PGP, mean (SD), No. of nerves/mm2 | 18.1 (6.6) | 20.3 (10.9) | 20.1 (7.3) | 28.3 (13.2) | 55.1 (9.3) | <.001 |
| CGRP, mean (SD), No. of nerves/mm2 | 12.2 (9.8) | 19.0 (8.3) | 19.2 (6.1) | 22.0 (9.2) | 41.7 (26.9) | <.001 |
| CGRP/PGP ratio, % | 67.1% | 93.6% | 95.5 | 77.8% | 75.6% | .121 |
|
Horizontal direction
| ||||
|---|---|---|---|---|
|
Regions
|
P value | |||
| CL | CC | CP | ||
| PGP, mean (SD), No. of nerves/mm2 | 26.9 (9.3) | 15.6 (7.3) | 7.1 (7.4) | .0025 |
| CGRP, mean (SD), No. of nerves/mm2 | 23.1 (7.6) | 12.0 (13.2) | 5.1 (5.4) | .0021 |
| CGRP/PGP ratio, % | 85.8% | 76.8% | 72.3% | .547 |
PGP, protein-gene product; CGRP, calcitonin gene-related peptide; CL, periodontal ligament third; CC, center third; CP, periosteal third.
Figure 3.
Vertical Nerve Distribution Density Analysis
The left panel presents a photograph of the tissue specimen with black lines and letters indicating the 5 vertical regions. The right panel shows bar graphs of mean distribution density values. There were significant differences in the distribution density of PGP-positive (all) and CGRP-positive (nociceptive) nerves (P = 9.012−7 and P = .0002, respectively). The nerve distribution density increased significantly toward the superior alveolar nerve from the alveolar crest. PGP, protein-gene product; CGRP, calcitonin gene-related peptide.
Analysis of the horizontal nerve distribution density showed that the mean distribution density of PGP-positive and CGRP-positive nerves significantly increased horizontally from subregion CP to subregion CL, moving from the periosteal side toward the periodontal ligament side (P < .0025; Table 2; Figure 4).
Figure 4.
Horizontal Nerve Distribution Density Analysis
The bottom panel shows a photograph of the tissue specimen with the 3 horizontal subregions represented by black line and letters. The top panel shows bar graphs corresponding to each region. There were significant differences in the mean distribution density of PGP-positive (all) and CGRP-positive (nociceptive) nerves (P = .0025 and P = .0021, respectively). The nerve distribution density increased significantly toward the periodontal ligament side. PGP, protein-gene product; CGRP, calcitonin gene-related peptide; CL, periodontal ligament third; CC, center third; CP, periosteal third.
Regarding the ratio of CGRP- to PGP-positive nerves, the ratio was greater than 80% on average (Table 2). There was no significant difference in the ratio of CGRP- to PGP-positive nerves in the vertical and horizontal regions (Table 2).
DISCUSSION
The normal human maxilla was not used for tissue specimens in this study because of serious ethical problems; therefore, we used the maxilla of rats to analyze nerve distribution density. The rat maxilla has frequently been used as an experimental model to imitate the human maxilla in anatomical, histological, and surgical studies. In addition, it is a highly reproducible, ideal histological model.10,11 Therefore, the nerve distribution density in the human maxilla can be inferred to a certain degree from our findings.
Nerve distribution densities in intraoral tissues such as the temporomandibular joint, the periodontal ligament, alveolar bone, and the dental pulp as well as some osseous tissues have been reported.12–15 However, the ratios of nerve distribution densities of different sites were not statistically analyzed in those reports. The present study reflects the original research of “Immunohistochemical analysis of nerve distribution in mandible of rats” by Kudo et al,5 which was previously reported by our department. In this study, the nerve distribution density of all nerves and of nociceptive nerves in the maxilla was measured using the same method employed by Kudo et al5 at the same facility under the same research director, and the ratios were then statistically analyzed and compared. Therefore, our data on the nerve distribution density in the maxilla can be compared with data from Kudo et al5 on nerve distribution density in the mandible.
According to our measurement results, the nerve distribution density in the maxilla increased significantly from the alveolar crest (region A) toward the superior alveolar nerve (region E) which is situated deeper. In other words, nerve distribution in the maxilla became denser in areas closer to the main branches and trunks of nerves, such as the superior alveolar nerve. This finding was similar to the study involving rat mandibles which found that nerve density increased significantly in a vertical direction moving from the alveolar crest toward the inferior alveolar nerve. However, the mean overall nerve distribution density in the vertical direction of the maxilla (28.4 nerves/mm2) was only approximately one-third of that of the mandible (80.5 nerves/mm2). In the horizontal direction, nerve distribution in both the maxilla and mandible was significantly denser in the periodontal ligament side than in the periosteal side. However, also in the horizontal direction, the mean overall nerve distribution of maxilla (16.5 nerves/mm2) was only approximately one-fifth that of the mandible (88.4 nerves/mm2).
In the maxilla, superior alveolar nerves branching from the maxillary nerve (the second division of the trigeminal nerve) run in the alveolar canals of the lateral and anterior walls of the maxillary sinus and anastomose with each other to form the superior alveolar nerve plexus.16 From the superior alveolar nerve plexus, the branches then innervate the maxillary teeth. Therefore, we speculate that the number of nerve branches increases in regions closer in proximity to the superior alveolar nerve.
The nerve distribution density increases significantly in the horizontal direction moving from the periosteal side to the periodontal ligament side. The periodontal ligament supports the teeth, provides a buffer against occlusal force, and is densely packed with sensory nerves. Because the periodontal ligament functions as a sensory organ for nociceptive and non-nociceptive sensory input, it contains many types of nerves as indicated by the previous findings of a high number of Ruffini endings,17 CGRP-positive nerves, and capillaries18 associated with the periodontal ligament. In the present study, nerve distribution became denser in areas closer to the periodontal ligament side. Thus, just like the mandible, nerve distribution in the maxilla is denser in regions closer in proximity to the periodontal ligament. Similar to the previous comparison, the overall nerve distribution in the horizontal direction also tended to be less dense in the maxilla as compared to the mandible.
Pain Sensation and Local Anesthesia in the Maxilla
The superior alveolar nerve in the maxilla is a somatosensory nerve and therefore a nociceptive nerve. Nociceptive nerve fibers are mainly composed of thick myelinated Aδ fibers and thin unmyelinated C fibers.19
In the present study, nerve fibers were stained using PGP and CGRP antibodies. Because the PGP antibody specifically stains cell bodies and axons in nerves, it stains all nerve fibers.7 The CGRP antibody specifically stains sensory nerves, and both Aδ and C fibers have synaptic vesicles containing CGRP. Therefore, CGRP antibodies can be used to specifically stain Aδ and C fibers.20,21 In analyzing the ratios of the mean distribution densities of nociceptive nerves to all nerves, we found that nociceptive nerves accounted for at least 80% of all nerves in the maxilla, similar to what was found in the mandible. These results indicate that sensory nerves, particularly nociceptive nerves, account for a large proportion of all nerves in both the maxilla and mandible and that sensory afferent nerves are also more common than voluntary motor nerves in both.
Local anesthesia in the maxilla22 is thought to be high successful because the maxillary cortical bone is more porous than the mandibular cortical bone and infiltration through the porous maxillary bone is therefore more efficient.23–25 According to a different report, local anesthesia via buccal infiltration anesthesia alone was sufficient for maxillary tooth extraction,26 which may suggest that local anesthesia infiltration is effective in the maxilla than in the mandible. The results of this study show that the nerve distribution density in the maxilla is only a third of that in the mandible. These findings may support future dental practice and research on local anesthetic effects and pain.
CONCLUSION
The nerve distribution density in the molar region of rat maxilla was measured and analyzed in both the vertical and horizontal directions. Similar to that in the mandible, the nerve distribution density in the maxilla significantly increased vertically in regions closer in proximity to the superior alveolar nerve and horizontally in regions closer in proximity to the periodontal ligament. The percentage of nociceptive nerves compared to total nerves was over 80%. However, the overall nerve distribution density in the maxilla was approximately one-third of that in the mandible. The results of this study describe the nerve distribution density in the maxilla which may support future research on pain and local anesthesia in the orofacial complex.
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