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Objective: Semaphorin 3A (Sema3A) is an essential chemorepellant controlling peripheral axon pathfinding and patterning, but also serves non-neuronal cellular functions. Incisors of rodent are distinctive from molars as they erupt continuously, have only one root and enamel is present only on the labial side. The aim of this study is to address putative regulatory roles of Sema3A chemorepellant in the development of incisor innervation and formation., Materials and Methods: This study analyzed expression of Sema3A mRNAs during embryonic and early post-natal stages of mouse mandibular incisor using sectional radioactive in situ hybridization., Results: Although Sema3A mRNAs were observed in condensed dental mesenchyme during the early bud stage, they were absent in dental papilla or pulp at later stages. Sema3A mRNAs were observed in the dental epithelium including the cervical loops and a prominent expression was also seen in alveolar bone. Interestingly, transcripts were absent from the mesenchymal dental follicle target area (future periodontal ligament) throughout the studied stages., Conclusion: The expression patterns of Sema3A indicate that it may control the timing and patterning of the incisor innervation. In particular, Sema3A appears to regulate innervation of the periodontal ligament, while nerve penetration into the incisor dental pulp appears not to be dependent on Sema3A. Moreover, Sema3A may regulate the functions of cervical loops and the development of alveolar bone. Future study with Sema3A deficient mice will help to elucidate the putative neuronal and non-neuronal functions of Sema3A in incisor tooth development.

Objective: Semaphorins form a family of axon wiring molecules but still little is known about their role in tooth formation. A class 3 semaphorin, Semaphorin3F (Sema3F), besides acting as a chemorepellant for different types of axons, controls a variety of non-neuronal developmental processes., Materials and Methods: Cellular mRNA expression patterns of Sema3F as well as neuropilin 1 (Npn1), neuropilin 2 (Npn2), plexinA3 and plexinA4 receptors were analyzed by sectional in situ hybridization in the mouse molar tooth during postnatal days 0-7. The expression of the receptors was studied in PN5 trigeminal ganglia., Results: Sema3F, Npn1, -2 and plexinA4 exhibited distinct, spatiotemporally changing expression patterns, whereas plexinA3 was not observed in the tooth germs. Besides being expressed in the base of the dental mesenchyme Sema3F, like plexinA4, Npn1 and -2, was present in the ameloblast cell lineage. Npn1 and Npn2 were additionally seen in the pulp horns and endothelial cells and like PlexinA4 in the developing alveolar bone. Npn1, plexinA3 and -A4 were observed in trigeminal ganglion neurons., Conclusions: Sema3F may act as a tooth target-derived axonal chemorepellant controlling establishment of the tooth nerve supply. Furthermore, Sema3F, like Npn1, -2 and plexinA4 may serve non-neuronal functions by controlling the development of the ameloblast cell lineage. Moreover, Npn1 and Npn2 may regulate dental vasculogenesis and, together with PlexinA4, alveolar bone formation. Further analyses such as investigation of transgenic mouse models will be required to elucidate in vivo signaling functions of Sema3F and the receptors in odontogenesis.

Objective: Our goal was to study the development of pioneer sympathetic innervation of dental pulp of mouse mandibular first molar., Design: We used double fluorescent immunohistochemistry with tyrosine hydroxylase (TH) and anti-medium-chain neurofilament (2H3) antibodies to detect sympathetic and sensory nerve fibres. Serial sections of whole teeth from postnatal days (PN) 0-14, trigeminal and sympathetic superior cervical ganglia of PN 15 mice were examined with confocal microscope., Results: There were two main findings. The unexpected finding was that 2H3 antibody was specific only for sensory nerve fibres and neurons and failed to stain either sympathetic nerve fibres or neurons. The main finding was that although both sympathetic and sensory nerve fibres were already seen near the tooth germ at the newborn stage, the pioneer sympathetic nerve fibres were first observed in the dental pulp only after the onset of root formation on day 9, in contrast to sensory nerve fibres which entered the tooth already on day 4., Conclusion: Pioneer sympathetic innervation of dental pulp starts on postnatal day 9 and follows sensory innervation. This indicates differential developmental regulation of the initial sensory and sympathetic innervation of teeth and provides essential background data for further studies on the molecular regulation of pulp innervation.

Substance P induces inflammatory reactions in peripheral tissues including the dental pulp, but its regulatory effects in target tissues are dependent on receptor signalling. Here the expression of the substance-P receptor neurokinin-1 (NK1) in the mature molar pulp of the rat was examined in order to localize the main target areas for substance P. A polyclonal antibody directed against the C-terminal of the receptor was used, and immunohistochemistry was performed by the avidin-biotin peroxidase complex method. The results showed that the NK1 receptor was intensely expressed along vessel-like structures in the odontoblast and subodontoblast layer. A granulated and diffusely distributed NK1-receptor labelling was found along larger blood vessels in the root pulp and pulp proper. NK1 receptor-positive cells were frequently observed in the cell-rich zone beneath the odontoblast layer. The results indicate that, in the mature rat molar pulp, the main targets for substance P acting through the NK1 receptors are tissues related to blood vessels in the odontoblast and subodontoblast area. Furthermore, the expression of NK1 receptors on cells located in the subodontoblast area could indicate that substance P also affects cell functions in this area.

The effect of this axotomy on the expression of vasoactive intestinal polypeptide (VIP) in trigeminal ganglion neurones and nerve fibres in the first molar was examined immunohistochemically 3 weeks postsurgically in rats. A distinct upregulation of VIP-like immunoreactivity was found in 3 to 4% of the neurones (mean dia., 20.9 +/- 0.45 microns; mean cross-sectional area, 367 +/- 13.2 microns 2) in the mandibular region after axotomy. An almost complete coexpression was registered in neurones upregulated for VIP and growth-associated protein 43. Coexpression of VIP and substance P (SP) was found in a small number of the immunoreactive (IR) small-sized neurones, mainly in those in which VIP and SP were both weakly immunoreactive. In the uninjured ganglion, weakly labelled VIP-positive granules were frequently traced at high magnification in the cytoplasm of small neurones. No VIP-IR fibres were found in the control molar pulp, except for a few in the root pulp near the apex. However, 3 weeks after axotomy, a number of VIP-containing nerve fibres were found in the molar pulp and apical periodontium. Coarse VIP-IR fibres directed towards the odontoblast layer were a common finding. In some of these fibres VIP was shown to colocalize with SP. These results indicate that VIP is clearly expressed and transported in regenerating primary afferent neurones after axotomy of the inferior alveolar nerve.

The aims of this study were to evaluate the effect of inferior alveolar nerve (IAN) axotomy on periodontal (PDL) and pulpal blood flow incident to experimental tooth movement and to investigate whether nerve fiber regeneration coincides with blood flow changes. The first right mandibular molar was moved mesially for 3, 7, and 14 days after ipsilateral IAN axotomy in 29 rats. Four rats served as unoperated controls. At the end of each experimental period fluorescent microspheres (FM) were injected into the left ventricle and thereafter counted in serial sections in the PDL and pulp of the right and left first mandibular molars. The number of FM per tissue volume was taken as a measure of blood flow. Re-innervation of nerve fibers was mapped immunohistochemically 7, 14, and 21 days after IAN axotomy in 9 rats that had no orthodontic appliance. The statistical analysis showed no significant differences in the number of FM/mm3 PDL between the denervated and the contralateral side at 3 and 7 days. At 14 days the PDL on the denervated side showed a significant increase in the number of FM/mm3, coinciding with the initial periodontal nerve fiber re-innervation. In the pulp no significant differences were found between the denervated and the contralateral, innervated side in any experimental period. It can be concluded that IAN axotomy postpones an increase in periodontal blood flow until a sensory tissue re-innervation is established, thus indicating that neurogenic mechanisms play an important role in the development of the inflammatory reaction induced by experimental tooth movement.

The effect of experimental tooth movement on nerve fibers immunoreactive to calcitonin gene-related peptide (CGRP) and to protein gene product (PGP) 9.5 were studied, as well as the coincidence of these responses with changes in blood vessel density and distribution in the periodontal ligament (PDL) and pulp of young Wistar rats. The first right maxillary molar was moved mesially by an orthodontic appliance for 3, 7, 14 and 21 days. Sagittal and horizontal serial sections were incubated alternately with antibodies to CGRP, PGP 9.5 and laminin. Nerve and blood vessel density and distribution between the experimental and control sides were compared in the apical and cervical PDL, and in root and coronal pulp. The most pronounced changes occurred in the 7 day group. CGRP and PGP 9.5 immunoreactive nerves in the apical PDL showed increased density, being distributed towards the alveolar bone and frequently found in bone resorption lacunae. Numerous nerve fibres were often present adjacent to hyalinized tissue, but were never found near or within root resorption lacunae. Nerve sprouting was also present both in the root and coronal pulp. Increased nerve and blood vessel density generally coincided with each other. At day 14, periodontal nerves and blood vessels were still disorganized compared with the controls. Tissues near cellular cementum and root resorption lacunae were consistently devoid of nerve fibres. After 21 days, PDL nerve and blood vessel density and distribution were nearly at control level. However, nerve fibres were regularly found inside root resorption areas. In conclusion, experimental tooth movement induces dynamic changes in density and distribution of periodontal and pulpal nerve fibres, indicating their involvement in both early stages of periodontal remodelling and later in the regenerative processes of the PDL, generally occurring in concerted action with modulation of blood vessels.

Release of sensory neuropeptides after stimulation of afferent nerve fibers has previously been shown to induce vasodilation and increased vascular permeability in the dental pulp, a condition recognized as neurogenic inflammation. In the present study a possible role for the sensory neuropeptides in transendothelial migration of immunocompetent cells was investigated. The dental pulp is an isolated tissue densely innervated with sensory fibers containing neuropeptides, and following electrical stimulation of the crown, the effect on pulpal blood flow and immunocompetent cells can be studied in a noninvasive model. A laser Doppler flowmeter was used to measure relative changes in pulpal blood flow during long-lasting intermittent stimulation of innervated and denervated rat first molars. In the innervated teeth, stimulation promptly increased pulpal blood flow by on average 45% at the start of the experiment, whereas almost no blood flow increase was recorded after 4 to 5 h stimulation. Surgical sectioning of the inferior alveolar nerve abolished blood flow increase upon stimulation. After stimulation, a quantitative analysis of CD43+, CD4+, CD11+, and I-A antigen-expressing cells was performed, and the effect of stimulation on calcitonin gene-related peptide (CGRP)-immunoreactive and substance P (SP)-immunoreactive (IR) nerve fibers was studied. Immunohistochemistry was performed by the avidin-biotin peroxidase method. Stimulation resulted in an almost complete depletion of CGRP- and SP-IR nerve fibers in the first molar pulp, whereas nerve fibers in the gingiva and neighboring teeth were unaffected. A significant increase in the number of CD43+ cells was found in the innervated tooth after stimulation compared to the stimulated denervated (P < 0.01) and unstimulated control (P < 0.05) first molars. For I-A antigen-expressing cells a significant increase (P < 0.05) was found between the innervated stimulated and unstimulated control, but not between the innervated and denervated stimulated first molars. Hence, from the present experiment it is concluded that the pulpal nerves participate in and facilitate transendothelial migration of CD43+ cells during acute neurogenic inflammation.

Denervation has been a useful approach to the investigation of interactions between nerve fibres and the pulp-dentine complex. Information on the immunological implications of axotomy is still lacking. The effect of axotomy on CD43+, CD4+, CD11b+ and I-A antigen-expressing cells in both the distal segment of the cut inferior alveolar nerve and in the first molar pulp of young rats was evaluated. Nerve fibres immunoreactive to protein gene product (PGP) 9.5, the neuropeptides substance P and calcitonin gene-related peptide (CGRP), and neuropeptide Y were visualized also by use of the avidin-biotin peroxidase complex method. Recruitment of macrophages was found in the distal segment of the sectioned inferior alveolar nerve 2 days after axotomy, with a further increase in number during the 6-day observation period. However, in the dental pulp, the number of CD43+, CD4+, CD11b+ and I-A antigen-expressing cells was almost unaffected. An almost complete sensory denervation of the first mandibular molar pulp was obtained 2 days after axotomy. After 6 days, the mesial part of the coronal pulp still remained denervated, while regenerated nerve fibres had reached both the root pulp and the distal part of the coronal pulp. Nerve fibres immunoreactive to neuropeptide Y were slightly reduced in density 2 days after axotomy, and after 6 days the localization of neuropeptide Y-immunoreactive fibres was changed compared to the control, with fibres also distributed in the odontoblast layer close to dentine. Hence, following axotomy in young rats, an almost complete sensory denervation is achieved in the first molar, whereas nerve fibres immunoreactive to neuropeptide Y change their distribution pattern, with fibres located close to the dentine after 6 days. Due to the almost unchanged number and distribution of immunocompetent cells in the pulp after axotomy, the young rat molar pulp may represent a suitable and useful experimental model to study neuro-immune interactions.

The dental pulp represents a peripheral end-organ deprived of a collateral nerve supply. After inferior alveolar nerve (IAN) axotomy, rat molar pulp is denervated over a period of at least 6 days. Therefore, rat molar pulp was used as an experimental model to study the effect of sensory nerve fibers on influx of immunocompetent cells after dentinal injury. In the present study we performed a quantitative analysis of CD43+, CD4+, CD11b+, and I-A antigen-expressing cells subjacent to dentinal cavities in denervated and innervated first mandibular molars. For visualization of nerve fibers, antibodies to protein gene product (PGP) 9.5, the sensory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP), and the sympathetic neuropeptide Y (NPY) were used. Immunohistochemistry was performed by the avidin-biotin-peroxidase method. In the innervated teeth, a correlation between increased sensory nerve density and influx of immunocompetent cells was found. Compared to the contralateral innervated molars, a significant reduction in recruitment of immunocompetent cells was found in the denervated pulp tissue subjacent to the dentinal cavities. The rat molar represents a unique model to illustrate the influence of sensory nerves and neuropeptides on inflammation and recruitment of immunocompetent cells.