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Fibroblast-like cells from rat plantar skin and neurotrophin-transfected 3T3 fibroblasts influence neurite growth from rat sensory neurons in vitro
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Division of Molecular Neurobiology, MBB, Karolinska Institute, Stockholm, Sweden.
Division of Molecular Neurobiology, MBB, Karolinska Institute, Stockholm, Sweden.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
2001 (English)In: Journal of Neurocytology, ISSN 0300-4864, E-ISSN 1573-7381, Vol. 29, no 9, 653-663 p.Article in journal (Refereed) Published
Abstract [en]

Our previous finding that skin-derived and muscle-derived molecules can be used to sort regenerating rat sciatic nerve axons evoked questions concerning neuron-target interactions at the level of single cells, which prompted the present study. The results show that dorsal root ganglion (DRG) neurons co-cultured with fibroblast-like skin-derived cells emit many neurites. These have a proximal linear segment and a distal network of beaded branches in direct relation to skin-derived cells. Electron microscopic examination of such co-cultures showed bundles of neurites at some distance from the target cells and single profiles closely apposed to subjacent cells. RNase protection assay revealed that cultivated skin-derived cells express nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). In co-cultures of DRG neurons and 3T3 fibroblasts overexpressing either of the neurotrophins produced by skin-derived cells the picture varied. NT-3 transfected 3T3 fibroblasts gave a growth pattern similar to that seen with skin-derived cells. Neurons co-cultured with mock-transfected 3T3 fibroblasts were small and showed weak neurite growth. In co-cultures with a membrane insert between skin-derived cells or 3T3 fibroblasts and DRG neurons few neurons survived and neurite growth was very sparse. We conclude that skin-derived cells stimulate neurite growth from sensory neurons in vitro, that these cells produce NGF, BDNF, NT-3 and NT-4 and that 3T3 fibroblasts producing NT-3 mimic the effect of skin-derived cells on sensory neurons in co-culture. Finally the results suggest that cell surface molecules are important for neuritogenesis.

Place, publisher, year, edition, pages
2001. Vol. 29, no 9, 653-663 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-25082DOI: 10.1023/A:1010883320683Local ID: 9512OAI: oai:DiVA.org:liu-25082DiVA: diva2:245408
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Factors influencing nerve growth in situ and in vitro
Open this publication in new window or tab >>Factors influencing nerve growth in situ and in vitro
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [sv]

Bakgranden till denna doktorsavhaodling är den problematik som uppstår efter en perifer nervskada, roreträdesvis i relation till handen eftersom den har så stor betydelse i vårt dagliga liv. En haod utan känsel fungerar dåligt och än sänne fungerar en handprotes. Problemet idag är inte att få nervtrådarna att växa efter en skada utan att få dem att växa rätt. Dagens mikrokirurgiska behandling av nervskador är mycket förfinad. Vi kan inte vänta oss att en fortsatt utveckling av mikrokirurgin på ett dramatiskt sätt skall bidra till en rorbättring av vår rormåga att leda utväxande nervtrådar till deras mål organ. Därror är det viktigt att studera de molekylära faktorer som bidrar till styrning av nervväxt För att studera detta har jag använt mig av två olika modeller. Först gjorde jag en experimentell studie på vuxen råtta. Frågeställningen var om man med lljälp av filtrat gjorda från två olika målorgan (muskel och hud) kan styra utväxaode nervtrådar att växa åt rätt håll. Detta visade sig vara möjligt, och frågan väcktes då hur samspelet mellao nerv och målorgao såg ut på cellnivå. För att besvara den frågan utvecklade jag en in vitro-modell där jag odlade känselnervceller tillsammans med bindvävsceller från huden (känselcellemas målorgan). Jag har använt denna odlingsmodell i tre arbeten ror att undersöka hur vissa kända molekyler påverkar nervväxt. I ett arbete beskriver jag effekter av lösta molekyler (neurotrofiner) på nervväxt I ett annat arbete studeras betydelsen av molekyler som är bundna till cellytor (celladhesionsmolekyler) för nervväxt. En tredje studie rör effekter av ämnen som flisätts vid inflammation (cytokiner) på nervväxt. Sammanfattningsvis har denna avhaodling bidragit till att öka vår kunskap om olika faktorer som är viktiga för nervväxt I en framtid kommer vi förhoppningsvis attkunna sätta ihop all den konskap om nervväxt som just nu ackumul eras på olika håll i världen till en aovändbar helhet och tillämpa den vid behaodlingen av perifera nervskador.

Abstract [en]

Since peripheral nerves extend over long distances and follow a partly superficial course they are often subjected to injuries. This is especially true for major nerves in the extremities. Axotomized neurons can regenerate the divided axon, provided that a distal stump is available. However, successful microsurgical anastomosis of the stumps does not mean that each regenerating axon grows into the appropriate band of Biingner and back to the relevant target. In fact, regeneration often results in a neuron/target mis-match and an unsatisfactory functional restoration. Erratic regeneration is a serious therapeutic problem which can not be solved by a refined microsurgery. To improve the precision of axonal regeneration the nerve fibers have to be guided to their target organ via siguals at the cellular level. The general aim of this thesis was to identify some factors of importance for neurite growth in situ and in vitro.

The results of axon sorting experiments and retrograde tracing showed that adult rat sciatic motor axons regenerating into a Y-tube grew more readily into a branch containing muscle-derived molecules than into a branch containing skin-derived molecules. Regenerating sciatic sensory axons emerging from large perikarya were emiched in a branch with muscle-derived molecules and sensory axons from small perikarya were enriched in a branch with skin-derived molecules.

Experiments in vitro showed that skin-derived fibroblast-like cells (sFLCs) stimulate neurite formation from young DRG neurons. The nemites formed terminal-like networks iu close relation to iudividual sFLCs. RPA-analysis showed that sFLCs cultivated iu vitro expressed NGF, BDNF, NT-3 and NT-4 and that DRG neurons eocultured with transfected 3T3 cells were iuflueuced by neurotrophlns produced by these cells. A picture similar to the wild-type pattern was seeu iu co-cultures with 3T3 cells overexpressing NT-3.

Cell surface molecules appeared to play ao importaot role in the control of neuritogenesis from DRG neurons eo-cultured with sFLCs. RT-PCR analysis showed that sFLCs expressed the adhesion factors N-CAM, L1, N-cadherin, and ninjurin if cultured alone and N-cadherin only if DRG extract was added to the culture. Denervated and innervated whole skin samples differed similarly. Application of antibodies showed that adhesion factors L1, N-cadherin and ninjurin are important for survival of and neuritogenesis from DRG neurons co-cultured with sFLCs.

It was also found that sFLCs and perichondrial (p) FLCs expressed receptors for the cytokines IL-1ß, IL-6, LIF and that these cytokines affected DRG neurons eocultivated with both FLC types, in tenns of survival and/or neuritogenesis. The cytokine effects on DRG neurons eo-cultured with FLCs were influenced by cytokine concentration and by the origin of the FLCs. Some cytokine effects were mediated via NGF.

Altogether, these results show that nerve growth can be experimentally influenced by target-derived molecules in situ and by neurotrophins, cell adhesion molecules and cytokines in vitro. In the future, these and other factors may conceivably be used as tools for treatment of nerve injuries.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2001. 92 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 693
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25667 (URN)10043 (Local ID)91-7219-988-1 (ISBN)10043 (Archive number)10043 (OAI)
Public defence
2001-11-02, Berzeliussalen, Universitetssjukhuset, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2015-09-18Bibliographically approved

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Jerregård, HelenaHildebrand, Claes

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