Neurites from trigeminal ganglion explants grown in vitro are repelled or attracted by tooth-related tissues depending on developmental stage.

Although neurite attracting factors are present in the developing dental pulp and trigeminal ganglion (TG) axons can respond to such factors, nerve fibres do not enter the tooth pulp until a late developmental stage compared with surrounding tissues supplied by the TG. This suggests that the dental pulp secretes neurite growth inhibitory molecules. Semaphorins represent one group of substances, which can inhibit/repel growing neurites. The aims of the present study were to investigate if dental tissue explants inhibit/repel neurite growth from TGs at some developmental stages in vitro, and if so, to seek evidence for or against a participation of semaphorins in that interaction. By co-culturing mandibular or dental epithelial and mesenchymal tissue explants and TGs in collagen gels, we found that embryonic day 11 (E11) mandibular and E13 dental mesenchymal explants repel neurites from corresponding TGs. Repulsion was replaced by attraction if tissues from late embryonic or early postnatal mice (E17-postnatal day 5) were used. Using semi-quantitative reverse transcription/polymerase chain reaction we showed that a number of semaphorins were expressed by tooth-related mesenchyme collected from embryonic and postnatal mice. The expression of some semaphorins (3A, 3C, 3F, 4F, 5B, 6A, 6B and 6C) was high early in development and then decreased in a temporal pattern that correlated with neurite inhibitory/repulsive effects of dental mesenchyme observed in co-cultures. The expression of other semaphorins increased with development (3B, 4A and 7A), whilst others varied irregularly or remained at a fairly constant level (3E, 4B, 4C, 4D, 4G and 5A). Immunohistochemistry was used to determine if tooth-related nerve fibres possess neuropilins. This revealed that axons surrounding embryonic tooth buds express neuropilin-1, but not neuropilin-2. In postnatal teeth, nerve fibres located within the tooth pulp were immunonegative for neuropilin-1 and neuropilin-2. We conclude that developing mandibular/dental mesenchyme can inhibit/repel neurite growth in vitro. Our results support the hypothesis that semaphorins may be involved in this interaction.

Responses of rat trigeminal neurones to dental pulp cells or fibroblasts overexpressing neurotrophic factors in vitro.

The adult dental pulp is innervated by sensory trigeminal axons and efferent sympathetic axons. Rat trigeminal ganglia extend neurites when co-cultivated in vitro with pulpal tissue explants, suggesting that pulpal cells secrete soluble molecules that stimulate the growth of trigeminal ganglion axons. In addition, cultured pulpal cells produce mRNAs for neurotrophins and glial cell line-derived neurotrophic factor-family members. These data suggest that neurotrophic factors are involved in the formation of a pulpal innervation. Here, we examine how pulpal cells and 3T3 fibroblasts overexpressing certain neurotrophic factors (nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, glial cell line-derived neurotrophic factor or neurturin) influence survival and growth of single trigeminal ganglion neurones in vitro in quantitative terms. The results show that most of the neurotrophic factor-overexpressing fibroblasts induce similar neuronal soma diameters, but higher survival rates and neurite lengths compared with pulpal cells. With respect to neurite growth pattern, trigeminal ganglion neurones co-cultured with fibroblasts overexpressing nerve growth factor develop a geometry that is most similar to that seen in co-cultures with pulpal cells. We conclude that none of the fibroblasts overexpressing neurotrophic factors can fully mimic the effects of pulpal cells on trigeminal ganglion neurones, and that nerve growth factor promotes a neurite growth pattern most similar to the picture seen in co-cultures with pulpal cells.

Expression of ErbB3, ErbB4, and neuregulin-1 mRNA during tooth development.

The receptor tyrosine kinases ErbB3 and ErbB4, which bind to various variants of neuregulin (NRG), play fundamental roles in neural development and in organs, which form through epithelial-mesenchymal interactions. Here, we demonstrate that NRG-1 and the receptors ErbB3 and ErbB4 are expressed locally during rodent tooth development. However, the mRNA expression patterns of ErbB3 and ErbB4 were distinctly different during odontogenesis. Examinations of teeth in genetically heart-rescued ErbB4-/- mice did not reveal any obvious deviation from the normal phenotype. The results suggest that ErbB3 and ErbB4 may participate in tooth morphogenesis. The specific interactions between NRG isoforms and ErbB receptors during this process remain to be determined.

Rat tooth pulp cells elicit neurite growth from trigeminal neurones and express mRNAs for neurotrophic factors in vitro.

Molecular factors control the developmental ingrowth of axons to the tooth pulp. Here we examine the ability of pulpal cells to induce neurite outgrowth from neonatal rat trigeminal neurones (TGNs) in vitro. We found that TGNs emitted neurites and formed networks of branches in relation to pulpal cells. Neurones co-cultured with a mixture of pulpal cells and 3T3 fibroblasts formed networks exclusively in relation to the pulpal cells. Cultivated pulpal cells and pulpal tissue produced mRNAs for all neurotrophins and members of the glial cell line-derived neurotrophic factor family. Hence, rat pulpal cells have neuritogenic effects on single TGNs in vitro, that may be associated with secretion of neurotrophic factors.

Molecular signaling and pulpal nerve development.

The purpose of this review is to discuss molecular factors influencing nerve growth to teeth. The establishment of a sensory pulpal innervation occurs concurrently with tooth development. Epithelial/mesenchymal interactions initiate the tooth primordium and change it into a complex organ. The initial events seem to be controlled by the epithelium, and subsequently, the mesenchyme acquires odontogenic properties. As yet, no single initiating epithelial or mesenchymal factor has been identified. Axons reach the jaws before tooth formation and form terminals near odontogenic sites. In some species, local axons have an initiating function in odontogenesis, but it is not known if this is also the case with mammals. In diphyodont mammals, the primary dentition is replaced by a permanent dentition, which involves a profound remodeling of terminal pulpal axons. The molecular signals underlying this remodeling remain unknown. Due to the senescent deterioration of the dentition, the target area of tooth nerves shrinks with age, and these nerves show marked pathological-like changes. Nerve growth factor and possibly also brain-derived neurotrophic factor seem to be important in the formation of a sensory pulpal innervation. Neurotrophin-3 and -4/5 are probably not involved. In addition, glial cell line-derived neurotrophic factor, but not neurturin, seems to be involved in the control of pulpal axon growth. A variety of other growth factors may also influence developing tooth nerves. Many major extracellular matrix molecules, which can influence growing axons, are present in developing teeth. It is likely that these molecules influence the growing pulpal axons.

[Schwann cells promote development and repair of nerve cells]. / Schwanncellen främjar utveckling och reparation av nervceller.

During early development Schwann cells have trophic effects on neurons with outgrowing axons. Later these cells are responsible for myelination and formation of nodes of Ranvier in the peripheral nervous system, a developmental process with considerable functional significance. In adult nerves, Schwann cells and axons cooperate closely. After nerve injuries, axons degenerate while Schwann cells proliferate and dedifferentiate. The stimulating effects these cells have on axonal regeneration are exploited clinically through the use of nerve grafts for repair. Schwann cells are used experimentally to enhance regeneration of axons in the central nervous system. Tomorrow this may be used clinically--an exciting development.

Hypoglycaemic neuropathy: occurrence of axon terminals in plantar skin and plantar muscle of diabetic BB/Wor rats treated with insulin implants.

It is generally believed that diabetic neuropathy is due to chronic hyperglycaemia. However, experience from insulinoma patients and experimental studies show that hypoglycaemia may also cause neuropathy. Accordingly, the plantar nerves of diabetic eu-/hypoglycaemic BB/Wor rats treated with insulin implants exhibit a distinct neuropathy. To what extent hypoglycaemic neuropathy affects axon terminals in skin and muscle is unknown. In the present study we examine the occurrence of epidermal axon profiles and the neuropeptide calcitonin gene-related peptide (CGRP) in plantar skin, and of end plate axon terminals in a plantar muscle of diabetic BB/Wor rats subjected to long periods of hypoglycaemia. The number of protein gene product-immunoreactive axon profiles was found to be normal in heel skin biopsy specimens from eu-/hypoglycaemic rats, but many profiles were short and thin. The content of CGRP in the skin biopsy samples was significantly below normal. After staining with antibodies against the vesicular acetylcholine transporter protein, the occurrence of end plate axon terminals was significantly reduced in sections from the flexor hallucis brevis muscle of eu-/hypoglycaemic rats. Moreover, the end plate axon terminals tended to be abnormally small in these rats. We conclude that the hypoglycaemic neuropathy seen in plantar nerve trunks of diabetic BB/Wor rats treated with insulin implants is accompanied by mild alterations in the epidermal innervation of plantar skin and a more obviously abnormal nerve terminal pattern in plantar muscle.

Denervation does not affect the growth of rat vibrissae.

This study examines the hypothesis that neural factors influence the growth of rat vibrissae. We divided the vibrissae in rows alpha-delta, 1 and 2 and examined their regrowth during the first complete growth period in normal and nerve-lesioned rats. The lesions used were denervation through neonatal capsaicin treatment, surgical sympathecomy in adult rats, neurectomy of the mandibular and buccal branches of the facial nerve in adult rats or division of the infraorbital nerve in adult rats. Normal vibrissae developed a length of 51.1 mm and a diameter of 178 microm (row alpha-delta), 44.1 mm and 181 microm (row 1) and 33.2 mm and 165 microm (row 2). In all experimental groups the examined vibrissae developed a normal final length and proximal diameter. This indicates that local nerves do not influence vibrissal growth to any major extent.

Tooth pulp tissue promotes neurite outgrowth from rat trigeminal ganglia in vitro.

The mammalian tooth pulp becomes innervated by nociceptive and sympathetic axons relatively late during development, when part of the root has formed. In the adult, regenerating axons from an injured tooth nerve or sprouting axons from uninjured nerves in the vicinity rapidly reinnervate denervated tooth pulps. These observations indicate that tooth pulp tissue can use molecular factors to attract pulpal axons from local nerve trunks. The present study examines the hypothesis that these factors include nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and glial cell line derived neurotrophic factor (GDNF). Explants of trigeminal ganglia from neonatal rat pups showed a distinct neurite outgrowth when co-cultured with pulpal explants collected from molar teeth of 12-day old pups, or after application of a pulpal extract. Control cultures, containing single ganglionic explants, or explants co-cultured with heat-treated pulpal tissue, exhibited a sparse neurite outgrowth. Exogenous NGF and/or GDNF, but not exogenous BDNF, stimulated neurite outgrowth from ganglionic explants. Unexpectedly, application of antibodies against NGF, BDNF and/or GDNF to co-cultures of ganglionic and pulpal explants did not inhibit neuritogenesis. Control experiments showed that IgG molecules readily penetrate the gel used for culture and that even very high concentrations of NGF and GDNF antibodies in combination failed to block neurite growth. On the basis of these data we suggest that other as yet unknown neurite-promoting factors might be present and active in TG/pulpal co-cultures.