Slit1 is specifically expressed in the primary and secondary enamel knots during molar tooth cusp formation.

The shape and diversity of the mammalian molar teeth is suggested to be regulated by the primary and secondary enamel knots, which are putative epithelial signaling centers of the tooth. In search of novel molecules involved in tooth morphogenesis, we analyzed mRNA expression of Slit1, -2 and -3, earlier characterized as secreted signals needed for axonal pathfinding and their two receptors Robo1 and -2 (Roundabout1 and -2) in the developing mouse first molar. In situ hybridization analysis showed that Slit1 mRNAs were expressed in the primary enamel knot of the bud and cap stage tooth germ and later the expression continued in the secondary enamel knots of the late cap and bell stage tooth. In contrast, expression of Slit2 and -3 as well Robo1, and -2 was largely restricted to mesenchymal tissue components of the tooth until the bell stage. At the late bud stage, however, Robo1 transcripts were evident in the primary enamel knot, and at the cap stage a pronounced expression was noted in the middle of the tooth germ covering the primary enamel knot and dental papilla mesenchyme. During the bell stage, Robo1 and Slit2 expression became restricted to the dental epithelia, while Slit3 continued in the dental mesenchyme. Prior to birth, Robo1 and -2 were co-localized in the predontoblasts. These results indicate that Slits and Robos display distinct, developmentally regulated expression patterns during tooth morphogenesis. In addition, our results show that Slit1 is the second known gene specifically located in the primary and secondary enamel knots.

Expression of class 3 semaphorins and neuropilin receptors in the developing mouse tooth.

The semaphorins are a large family of secreted or cell-bound signals needed for the development of the nervous system. We compared mRNA expression of class 3 semaphorins (Sema3A, 3B, 3C and 3F) and their two receptors (Neuropilin-1 and -2) in the embryonic mouse first molar tooth germ (E10-18) by radioactive in situ hybridization. All genes showed distinct developmentally regulated expression patterns during tooth organogenesis. Interestingly, Sema3A and 3C were first detected in the early dental epithelium, and later both genes were present in the epithelial primary enamel knot, a putative signaling center of the embryonic tooth regulating tooth morphogenesis. Prior to birth, Sema3A was also observed in tooth-specific cells, preodontoblasts, which later differentiate into odontoblasts secreting dentin, and in the mesenchymal dental follicle cells surrounding the tooth germ. Sema3B appeared transiently in the dental mesenchyme in the bud and cap stage tooth while Sema3F was expressed in both epithelial and mesenchymal components of the tooth. Of note, Npn-1 expression pattern was largely complementary to that of Sema3A, and transcripts were restricted to the dental mesenchymal cells. Npn-1 expression was first seen in the developing dental follicle, and later transcripts also appeared in the dental papilla mesenchyme. In contrast, Npn-2 signal was seen in both epithelial and mesenchymal tissues such as in the primary enamel knot and preodontoblasts.

Neuroendocrine cells in Malassez epithelium and gingiva of the cat.

Malassez epithelium has been designated as epithelial cell rests, the biological significance of which is still under debate. This study was designed to analyze Malassez epithelium for the presence of neuroendocrine cells. Gingival tissue was included as a positive control. Using immunohistochemistry, confocal and light microscopy, Malassez epithelium and gingival epithelium from mature cats (n = 5) were examined for cells containing the neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP), and vasoactive intestinal peptide (VIP). Both Malassez epithelium and the basal epithelial cell layers in gingival rete pegs regularly displayed cells immunoreactive to CGRP, SP, and VIP. The immunopositive cells were most frequently present in the epithelial cell clusters and strands of Malassez located in the cervical half of thc periodontal ligament. Double immunolabeling revealed cellular co-expression of CGRP or SP with VIP, and the neuropeptides were co-localized in the cellular compartments. Labeled cells in both epithelia were occasionally supported by immunoreactive nerve fibers. This study shows that cells immunoreactive to CGRP, SP, and VIP arc located within the cat Malassez epithelium. The localization of neuroendocrine cells verifies the diversity of this epithelium and confirms that Malassez epithelium is composed of different cell types, in common with epithelia from other locations. The presence of neuroendocrine cells in Malassez epithelium strongly suggests biological functions of this tissue, and the neuropeptide content may thus indicate endocrine functions of the cells.

Neurokinin-1 receptor expression in the mature dental pulp of rats.

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.

Coexpression of vasoactive intestinal polypeptide and substance P in reinnervating pulpal nerves and in trigeminal ganglion neurones after axotomy of the inferior alveolar nerve in the rat.

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.

Effect of inferior alveolar nerve axotomy on periodontal and pulpal blood flow subsequent to experimental tooth movement in rats.

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.

Upregulation of growth associated protein 43 expression and neuronal co-expression with neuropeptide Y following inferior alveolar nerve axotomy in the rat.

Growth associated protein 43 (GAP 43) is an acidic membrane-bound phosphoprotein produced at high levels in developing and regenerating neurons. It is a substrate for protein kinase C and suggested to be involved in calcium-regulated release of axonal vesicular-contained neurotransmitters. Expression of GAP 43 has been demonstrated in the uninjured cat dental pulp which receives its sensory nerve supply from the trigeminal ganglion. The aim of this study was a detailed mapping of the spatial and time-dependent expression of GAP 43 and co-expression of neuropeptide Y (NPY) in dental peripheral target tissues and trigeminal neurons subsequent to inferior alveolar nerve (IAN) axotomy in rats, as background for later low-level laser studies. Unilateral sectioning of IAN, resulting in an almost complete loss of sensory nerve fibers in the ipsilateral dental pulp of the first molar, was performed. The avidin biotin complex (ABC) method was used to evaluate peripheral changes in GAP 43 expression at 4, 7 and 10 days. Ganglionic changes in GAP 43 and co-localization of neuronal NPY expression was examined at 4, 10 and 21 days using either the ABC method or double immunofluorescence labelling techniques and confocal microscopy. Axotomy resulted in an early upregulation and change in the peripheral distribution of GAP 43 in nerve profiles already 4 days post IAN axotomy suggesting a Schwann cell origin. Ten days post axotomy a pronounced upregulation of GAP 43 immunoreactivity could be demonstrated in neurons located in the mandibular region of the trigeminal ganglion, compared to the contralateral uninjured side. The peripheral and ganglionic upregulation of GAP 43 continued to persist at 21 days. A concomitant time-delayed shift and co-expression of NPY was demonstrated throughout in the GAP 43-upregulated ganglion cells 10 days post axotomy. Furthermore, confocal microscopy indicated that the intraneuronal distribution of NPY and upregulated GAP 43 expression showed a similar conformity and distribution in both perinuclear regions and cell periphery.

Effect of experimental tooth movement on nerve fibres immunoreactive to calcitonin gene-related peptide, protein gene product 9.5, and blood vessel density and distribution in rats.

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.

Effect of intermittent long-lasting electrical tooth stimulation on pulpal blood flow and immunocompetent cells: a hemodynamic and immunohistochemical study in young rat molars.

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.

Immunocompetent cells in rat periodontal ligament and their recruitment incident to experimental orthodontic tooth movement.

The aims of this study were to evaluate the number and distribution of immunocompetent cells in normal rat periodontal ligament (PDL) and to quantify their recruitment incident to experimental tooth movement. 27 young animals had the 1st right maxillary molar moved mesially by an orthodontic appliance for 1, 3, 7 and 14 days, respectively. 4 animals served as untreated controls. An immunohistochemical procedure was carried out on alternate serial cryostat sections, and monoclonal antibodies against CD11b (macrophages, dendritic cells), CD43 (lymphocytes, polymorphs), CD4 (helper T-lymphocytes), and class II MHC molecules were used. Mean counts of the immunolabeled cells in the control group showed the highest number of CD11b+ and class II molecule expressing cells, while CD4+ and CD43+ cells were scarcely found. Significant increase in the number of CD11b+, CD43+ cells and class II molecules was found in the PDL of the experimentally moved 1st molars compared with the contralateral side and the control group, while CD4+ cells showed no significant increase. CD11b+ and cells expressing class II molecules were found around hyalinized tissue, between dentin and cellular cementum and close to Malassez' epithelial cells. In conclusion, normal rat PDL has high number of macrophage and dendritic-like cells, but few lymphocytes and granulocytes. Furthermore, experimental tooth movement leads to significant recruitment of cells belonging to the mononuclear phagocytic system, but has no significant effect on the number of lymphocytes and granulocytes in the rat PDL.

Neuropeptide Y expression in the trigeminal ganglion and mandibular division of the trigeminal nerve after inferior alveolar nerve axotomy in young rats.

Neuropeptide Y (NPY) is a 36-amino-acid peptide residing in sympathetic nerve terminals, originating from the superior cervical ganglion in oral tissues. NPY exerts vasoconstrictor action together with noradrenalin and has been found to inhibit the release of neurotransmitters from primary afferent fibers. During regeneration of the axotomized inferior alveolar nerve (IAN), NPY-immunoreactive (IR) nerve fibers have been shown in the odontoblast layer and dentin, an area normally innervated by afferent nerve fibers. The dynamic shift in neuropeptide expression in the trigeminal ganglion and in the dental pulp was studied by immunohistochemistry 1, 2, 3, and 8 weeks after IAN axotomy. In the ipsilateral first mandibular molar a temporal loss of pulpal sensory nerves lasting for approximately 1 week was found after axotomy. An upregulation of NPY was shown in neurons located in the mandibular area of the trigeminal ganglion, concomitant to a reduction in number of neurons expressing substance P (SP). To study an alternate and possible trigeminal origin of some of the peripheral nerve fibers IR to NPY in the dental pulp, double immunofluorescence labeling was performed for NPY and calcitonin gene-related peptide (CGRP). Coexistence of NPY and CGRP was shown in neurons located in the trigeminal ganglion and in nerve fibers in the tooth pulp during IAN regeneration. Furthermore, retrograde tracing with Fluorogold revealed NPY-IR neurons projecting to the first molar pulp 3 weeks after axotomy. Hence, we conclude that after IAN axotomy NPY is produced in trigeminal ganglion neurons and transported in afferent regenerating fibers to the dental pulp. These results add further evidence for a plasticity in peptide transcription in sensory neurons after nerve injury and indicate a trigeminal origin of at least some of the pulpal NPY-IR fibers during nerve regeneration.

Effect of inferior alveolar nerve axotomy on immune cells and nerve fibres in young rat molars.

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.

Recruitment of immunocompetent cells after dentinal injuries in innervated and denervated young rat molars: an immunohistochemical study.

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.