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Sorting of Regenerating Rat Sciatic Nerve Fibers with Target-Derived Molecules
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
2001 (English)In: Experimental Neurology, ISSN 0014-4886, E-ISSN 1090-2430, Vol. 169, no 2, 298-306 p.Article in journal (Refereed) Published
Abstract [en]

The functional outcome of microsurgical repair of divided nerves is disappointing since many regenerating axons fail to reach appropriate targets. Sorting of regenerating axons according to target tissue might be used to improve functional regeneration. The aim of the present study is to see if regenerating axons can be sorted into functionally different bundles with target-derived molecules. The proximal stump of the adult rat sciatic nerve was sutured into the inlet of a silicon Y-tube. The two branches of the Y-tube were filled with agarose primed with filtrates prepared from skin and muscle homogenates from the operated rat. The tibial and sural nerves were inserted in the two branches of the Y-tube. Six weeks later the sciatic nerve axons showed vigorous regeneration into both branches. Electron microscopic examination of regenerated nerve segments showed numerous myelinated and unmyelinated axons. The proportion of myelinated axons was significantly larger in the muscle-gel branch than in the skin-gel branch. Retrograde tracing from the nerve regenerates with Fast Blue and Fluoro-Ruby showed that ventral horn neurons at L4–L5 segmental levels were preferentially labeled from the muscle-gel branch. Neurons in corresponding dorsal root ganglia were labeled from both Y-tube branches (no significant numerical difference). A few neurons of both types contained both tracers. Measurements revealed that sensory neurons labeled from the muscle-gel branch were significantly larger (mean perikaryal area 870 μm2) than neurons labeled from the skin-gel branch (mean area 580 μm2). We conclude that regenerating motor and sensory axons can be sorted with target-derived molecules.

Place, publisher, year, edition, pages
2001. Vol. 169, no 2, 298-306 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-25080DOI: 10.1006/exnr.2001.7656Local ID: 9510OAI: oai:DiVA.org:liu-25080DiVA: diva2:245406
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2015-09-18Bibliographically 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, HelenaNyberg, TobiasHildebrand, Claes

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