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Molecular factors influencing nerve growth: studies on the developing rodent trigeminal ganglion and tooth pulp
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The adult tooth pulp has a rich protective sensory innervation from the trigeminal ganglion (TG). The developmental establishment of this exclusively nociceptive innervation is not fully understood today. However, an increasing amount of data indicates that the tooth pulp cells produce molecular factors, which attract relevant nerve fibres from adjacent major nerve trunks. Presently, a wide variety of agents, which influence the growth of nerve fibres in various contexts, are known. Molecular factors, which might influence the innervation process of the developing tooth, are the focus of this thesis.

Based on gene-expression studies it has been suggested that nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) may be involved in the formation of a pulpal innervation. However, these studies did not examine how pulpal tissue/pulpal cells and TGs/TG neurones actually interact in vitro. In the first of the four studies included in this thesis we show that pulpal explants from neonatal rat pups elicit neurite growth from eo-cultured TG explants. It was also fotmd that application of exogenous NGF or GDNF, but not BDNF, to single TGs in vitro gives a rich neurite outgrowth. However, treatment of TG/pulpal co-cultures with antibodies against these neurotrophic factors does not fully block the neuritogenic effect of the pulpal explant. This seems to suggest that other, as yet tm.known factors are also involved. The second paper supplements the first one by illustrating the pulpal/TG interaction at the level of single cells. The results show that single TG neurones emit relatively linear neurites up to a single pulpal cell or a group of such cells. Upon reaching the pulpal cell/cells the neurite changes behaviour and forms a network of thin beaded branches in direct relation to the cell/cells. In co-cultures of TG neurones and a mixture of pulpal cells and 3T3 fibroblasts the neurones form neuritic networks exclusively in relation to the former. Finally, RT/PCR analysis revealed that both pulpal tissue and cultured pulpal cells express the genes for all known mammalian neurotrophins and GDNF-family members. The third paper compares the effects of cultured pulpal cells, and transfected 3T3 fibroblasts overexpressing selected neurotrophic factors on single TG neurones in terms of survival and neurite growth. It was found that the different 3T3 fibroblasts affect the neurones in different ways, and that none of them fully mimic the influences of wild-type pulpal cells. Also, the different 3T3 fibroblasts induce dissimilar neurite branching patterns. The pattern associated with NGF-overexpressing 3T3 cells is most similar to the one seen in co-cultures with TG neurones and pulpal cell.

Onset of nerve growth to the tooth pulp occurs comparatively late in development, although tooth pulp cells produce neurite growth stimulating molecular factors already during embryonic development, and in spite of the fact that nerve fibres from neurones expressing the appropriate receptor genes are located in the vicinity. These observations suggested the hypothesis that axon growth to prospective tooth pulps is transiently actively blocked during embryonic and early neonatal development. But, when amelogenesis and dentinogenesis have commenced nerve fibres enter the tooth pulp. This indicates that the hypothetical blockade decreases after birth, allowing an innervation of the tooth pulp to become established. In order to test this hypothesis, tooth-related tissue and TG explants from mice at various developmental stages were eo-cultured. It was found that mesenchymal tissue collected from early developing jaw or tooth primordia repel TG neurites, and that corresponding tissue from teeth in a more advanced stage of development attract TG neurites. One group of molecules with neurite growth-inhibiting properties are the semaphorins. Hence, we examined the presence of mRNAs for selected semaphorins in mesenchymal tissue collected from the jaw or from tooth pulps at different developmental stages. The results show that some of the selected semaphorins are expressed in a temporal pattern that matches the observed neuriteinhibiting effects seen in vitro and that conforms with the idea that there is an early pulpalderived blockage of nerve fibre growth in situ. Also, it was found that nerve fibres in the vicinity of the early developing tooth are immunoreactive for a relevant semaphorin receptor, neuropilin-1. After birth, when amelogenesis and dentinogenesis have commenced, nerve fibres in the tooth pulp are immunonegative for that receptor.

To conclude, our results show that pulpal tissue/pulpal cells can influence nerve fibres from TG explants/TG neurones in vitro. Also, we provide evidence that neurotrophins, GDNF-family members and semaphorins may contribute to this influence.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2003. , 86 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 823
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-25679Local ID: 10055ISBN: 91-7373-514-0 (print)OAI: oai:DiVA.org:liu-25679DiVA: diva2:246227
Public defence
2003-11-21, Elsa Brändströmsalen, Hälsouniversitetet, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-17Bibliographically approved
List of papers
1. Tooth pulp tissue promotes neurite outgrowth from rat trigeminal ganglia in vitro
Open this publication in new window or tab >>Tooth pulp tissue promotes neurite outgrowth from rat trigeminal ganglia in vitro
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1999 (English)In: Journal of Neurocytology, ISSN 0300-4864, E-ISSN 1573-7381, Vol. 28, no 8, 663-670 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-49702 (URN)10.1023/A:1007008815621 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-12Bibliographically approved
2. Rat tooth pulp cells elicit neurite growth from trigeminal neurones and express mRNAs for neurotrophic factors in vitro
Open this publication in new window or tab >>Rat tooth pulp cells elicit neurite growth from trigeminal neurones and express mRNAs for neurotrophic factors in vitro
2001 (English)In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 308, no 3, 161-164 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25005 (URN)10.1016/S0304-3940(01)01997-8 (DOI)9426 (Local ID)9426 (Archive number)9426 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
3. Responses of rat trigeminal neurones to dental pulp cells or fibroblasts overexpressing neurotrophic factors in vitro
Open this publication in new window or tab >>Responses of rat trigeminal neurones to dental pulp cells or fibroblasts overexpressing neurotrophic factors in vitro
2003 (English)In: Neuroscience, ISSN 0306-4522, E-ISSN 1873-7544, Vol. 119, no 2, 443-451 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25081 (URN)10.1016/S0306-4522(02)00938-7 (DOI)9511 (Local ID)9511 (Archive number)9511 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
4. Neurites from trigeminal ganglion explants grown in vitro are repelled or attracted by tooth-related tissues depending on developmental stage
Open this publication in new window or tab >>Neurites from trigeminal ganglion explants grown in vitro are repelled or attracted by tooth-related tissues depending on developmental stage
2004 (English)In: Neuroscience, ISSN 0306-4522, E-ISSN 1873-7544, Vol. 125, no 1, 149-161 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
BDNF, Brain-derived neurotrophic factor, E, EDTA, Embryonic day, Ethylenediamine- tetraacetic acid, GAPDH, GDNF, Glial cell line-derived neurotrophic factor, Glyceraldehyde 3-phosphate dehydrogenase, Mouse, Neuropilin, RT/PCR, Semaphorin, Tissue culture, Tooth pulp
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-45772 (URN)10.1016/j.neuroscience.2004.01.008 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13Bibliographically approved

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