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Effects of IL-1β, IL-6 or LIF on rat sensory neurons co-cultured with fibroblast-like cells
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
2002 (English)In: Journal of Neuroscience Research, ISSN 0360-4012, E-ISSN 1097-4547, Vol. 67, no 2, 255-263 p.Article in journal (Refereed) Published
Abstract [en]

Inflammation may affect the local presence of sensory nerve fibers in situ and inflammatory mediators influence sensory neurons in vitro. In the present study we have investigated effects of the cytokines interleukin-1β (IL-1β, interleukin-6 (IL-6), and leukemia inhibitory factor (LIF) on survival of and neurite growth from neonatal rat sensory neurons co-cultured with fibroblast-like cells prepared from neonatal rat skin (sFLCs) or perichondrium (pFLCs). The results showed that both FLC types expressed receptors for all three cytokines. Five ng/ml of either cytokine, but not lower or higher concentrations, supported survival of DRG neurons co-cultured with sFLCs. Neuronal survival was also enhanced by addition of the soluble IL-6 receptor (rsIL-6R) with or without IL-6. In co-cultures with pFLCs neuronal survival was promoted by IL-6, increasing with cytokine concentration. Addition of rsIL-6R without IL-6 did also stimulate neuronal survival. The growth of neurites from DRG neurons co-cultured with sFLCs was stimulated by 0.5 ng/ml LIF, unaffected by 5 ng/ml LIF and inhibited by 50 ng/ml LIF. Considering DRG neurons co-cultured with pFLCs, 50 ng/ml of either of the three cytokines, as well as rsIL-6R conditioned medium, stimulated neurite outgrowth. Some of the cytokine effects observed were reduced by application of antibodies against nerve growth factor (NGF). We conclude that that the cytokines examined affect DRG neurons in terms of survival or neuritogenesis, that the effects are influenced by cytokine concentration and the origin of the FLCs and that some of the effects are indirect, probably being mediated by factors released from FLCs.

Place, publisher, year, edition, pages
2002. Vol. 67, no 2, 255-263 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-24998DOI: 10.1002/jnr.10092Local ID: 9418OAI: oai:DiVA.org:liu-24998DiVA: diva2:245322
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
2. Sensory nerve fibres, neuropeptides and cartilage: Experimental studies in the rat
Open this publication in new window or tab >>Sensory nerve fibres, neuropeptides and cartilage: Experimental studies in the rat
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

During development, maintenance and repair after injury, reciprocal interactions occur between the peripheral nervous system and the target tissues. In the Papers presented in this thesis, different aspects of such netvetarget influences between peripheral nerve fibres and skeletal tissues dtuing development and repair have been investigated in the rat. Developing rat cartilaginous bone primordia have a richly innervated and vascularised perichondriwn. In addition, larger bones exhibit cartilage canals containing blood vessels and putative sensory nerve fibres. Tills evoked the question if there is a nervous regulation of skeletal development. Denervation of the hind paws of young rats resulted in a deficient length growth but had no influence on the progress of secondary ossification. Since growth is mainly due to events in cartilage, cartilage projecting sensory neurones were identified and examined. Sensory neurones projecting to the rat cartilaginous distal femoral epiphyses were located mainly in the dorsal root ganglia (DRG) L3 and L4 and exhibited small or medium-sized diameters. A large proportion of these neurones contained the neuropeptides CGRP and/or SP. However, application of CGRP to cartilage explants in vitro did not stimulate the chondrocytes in terms of an elevation of the level of cyclic AMP. Another possibility would be that the neuropeptides affect the developmental growth of bone and chondrocytes indirectly via effects on the blood vessels. Experiments .involving tracing as above and eo-culture of labelled DRG neurones and perichondrial cells in combination with immunohistochenllstty or electrophysiology showed that the traced cultured neurones contained CGRP and/or SPin in vivo-like proportions and that most of the cartilage-projecting neurones were proton sensitive, This prompted the suggestion that the nerve fibres in the perichondrium and in cartilage canals might release CGRP and SP in response to local tissue acidosis, thereby promoting tissue homeostasis by monitoring the balance between vascular supply and metabolic load and by influencing angiogenesis and blood flow. Subsequently, possible target influences on the local presence of perichondrial sensory nerve fibres were investigated. Application of inflammation related cytokines (IL-1ß, IL-6 and LIF) affected sensory neurones eo-cultured with perichondrium- or skin-derived fibroblast-like cells in terms of survival and neurite growth. These effects were strongly influenced by the origin of the target cells. Finally, experiments using the adult rat patella showed that osteochondral defects heal spontaneously but incompletely and that healing is not accompanied by an increase of local nerve fibres at the times examined. In conclusion, the present results indicate that cartilagerelated sensory nerve fibres influence skeletal growth, that a high proportion of these neurones contain CGRP and SP, that CGRP does not activate chondrocytes in cartilage slices, that many cartilage related sensory nerve fibres are proton-sensitive· and likely have a vasoregulatory role, that inflammatory mediators have distinct effects on sensory neurones eo-cultivated with perichondrial cells and that healing of an osteochondral defect in the rat patella does not involve a local increase of cartilage-related nerve fibres.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2001. 70 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 712
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25639 (URN)10014 (Local ID)91-7373-141-1 (ISBN)10014 (Archive number)10014 (OAI)
Public defence
2001-12-14, Berzeliussalen, Universitetssjukhuset, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-08-23Bibliographically approved

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Edoff, KarinJerregård, Helena

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