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  • 1.
    Edoff, Karin
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Jerregård, Helena
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Effects of IL-1β, IL-6 or LIF on rat sensory neurons co-cultured with fibroblast-like cells2002In: Journal of Neuroscience Research, ISSN 0360-4012, E-ISSN 1097-4547, Vol. 67, no 2, p. 255-263Article in journal (Refereed)
    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.

  • 2.
    Jerregård, Helena
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Factors influencing nerve growth in situ and in vitro2001Doctoral thesis, comprehensive summary (Other academic)
    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.

    List of papers
    1. Sorting of Regenerating Rat Sciatic Nerve Fibers with Target-Derived Molecules
    Open this publication in new window or tab >>Sorting of Regenerating Rat Sciatic Nerve Fibers with Target-Derived Molecules
    2001 (English)In: Experimental Neurology, ISSN 0014-4886, E-ISSN 1090-2430, Vol. 169, no 2, p. 298-306Article 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.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-25080 (URN)10.1006/exnr.2001.7656 (DOI)9510 (Local ID)9510 (Archive number)9510 (OAI)
    Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
    2. Fibroblast-like cells from rat plantar skin and neurotrophin-transfected 3T3 fibroblasts influence neurite growth from rat sensory neurons in vitro
    Open this publication in new window or tab >>Fibroblast-like cells from rat plantar skin and neurotrophin-transfected 3T3 fibroblasts influence neurite growth from rat sensory neurons in vitro
    2001 (English)In: Journal of Neurocytology, ISSN 0300-4864, E-ISSN 1573-7381, Vol. 29, no 9, p. 653-663Article 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.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-25082 (URN)10.1023/A:1010883320683 (DOI)9512 (Local ID)9512 (Archive number)9512 (OAI)
    Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
    3. Sensory neurons influence the expression of cell adhesion factors by cutaneous cells in vitro and in vivo
    Open this publication in new window or tab >>Sensory neurons influence the expression of cell adhesion factors by cutaneous cells in vitro and in vivo
    2001 (English)In: Journal of Neurocytology, ISSN 0300-4864, E-ISSN 1573-7381, Vol. 30, no 4, p. 327-336Article in journal (Refereed) Published
    Abstract [en]

    Dorsal root ganglion (DRG) neurons co-cultured with skin-derived fibroblast-like cells (FLCs) show a strong neurite outgrowth. However, when physical contact between FLCs and neurons is prevented with membrane inserts, the DRG neurons exhibit a low survival and a deficient neurite growth. This indicates that cell adhesion molecules influence neuronal survival and neurite growth in co-cultures. The aim of the present study is to find out if selected adhesion molecules are expressed by cultivated FLCs with and without nervous influences, and/or by normal and denervated whole skin. RT-PCR data show that cultured FLCs and denervated skin express L1, N-CAM, N-cadherin and ninjurin, but not neurofascin or TAG-1. However, cultured FLCs exposed to DRG homogenates and innervated skin express N-cadherin only. Following application of neutralizing L1-, N-cadherin- and ninjurin-antibodies (but not N-CAM-antibodies) in the culture medium the mean number of surviving neurons is decreased. Co-cultures incubated with L1-, N-cadherin- or ninjurin-antibodies all show significantly less neurite outgrowth compared to controls. In conclusion, the findings in this paper indicate (i) that FLCs cultured in vitro and denervated whole skin express the cell adhesion factors L1, N-CAM, N-cadherin and ninjurin, (ii) that FLCs treated with neural molecules and innervated whole skin express N-cadherin only, (iii) that L1, N-cadherin and ninjurin are important for DRG neurons co-cultured with FLCs in vitro in terms of survival and neurite extension and (iv) that there may exist subpopulations of DRG-neurons with different sensitivities for N-cadherin- and ninjurin-antibodies.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-25115 (URN)10.1023/A:1014460413884 (DOI)9548 (Local ID)9548 (Archive number)9548 (OAI)
    Note

    On the day of the defence day the status of this article was accepted.

    Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
    4. Effects of IL-1β, IL-6 or LIF on rat sensory neurons co-cultured with fibroblast-like cells
    Open this publication in new window or tab >>Effects of IL-1β, IL-6 or LIF on rat sensory neurons co-cultured with fibroblast-like cells
    2002 (English)In: Journal of Neuroscience Research, ISSN 0360-4012, E-ISSN 1097-4547, Vol. 67, no 2, p. 255-263Article 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.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-24998 (URN)10.1002/jnr.10092 (DOI)9418 (Local ID)9418 (Archive number)9418 (OAI)
    Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
  • 3.
    Jerregård, Helena
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Sensory neurons influence the expression of cell adhesion factors by cutaneous cells in vitro and in vivo2001In: Journal of Neurocytology, ISSN 0300-4864, E-ISSN 1573-7381, Vol. 30, no 4, p. 327-336Article in journal (Refereed)
    Abstract [en]

    Dorsal root ganglion (DRG) neurons co-cultured with skin-derived fibroblast-like cells (FLCs) show a strong neurite outgrowth. However, when physical contact between FLCs and neurons is prevented with membrane inserts, the DRG neurons exhibit a low survival and a deficient neurite growth. This indicates that cell adhesion molecules influence neuronal survival and neurite growth in co-cultures. The aim of the present study is to find out if selected adhesion molecules are expressed by cultivated FLCs with and without nervous influences, and/or by normal and denervated whole skin. RT-PCR data show that cultured FLCs and denervated skin express L1, N-CAM, N-cadherin and ninjurin, but not neurofascin or TAG-1. However, cultured FLCs exposed to DRG homogenates and innervated skin express N-cadherin only. Following application of neutralizing L1-, N-cadherin- and ninjurin-antibodies (but not N-CAM-antibodies) in the culture medium the mean number of surviving neurons is decreased. Co-cultures incubated with L1-, N-cadherin- or ninjurin-antibodies all show significantly less neurite outgrowth compared to controls. In conclusion, the findings in this paper indicate (i) that FLCs cultured in vitro and denervated whole skin express the cell adhesion factors L1, N-CAM, N-cadherin and ninjurin, (ii) that FLCs treated with neural molecules and innervated whole skin express N-cadherin only, (iii) that L1, N-cadherin and ninjurin are important for DRG neurons co-cultured with FLCs in vitro in terms of survival and neurite extension and (iv) that there may exist subpopulations of DRG-neurons with different sensitivities for N-cadherin- and ninjurin-antibodies.

  • 4.
    Jerregård, Helena
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Nyberg, Tobias
    Linköping University, Department of Biomedicine and Surgery. Linköping University, Faculty of Health Sciences.
    Hildebrand, Claes
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Sorting of Regenerating Rat Sciatic Nerve Fibers with Target-Derived Molecules2001In: Experimental Neurology, ISSN 0014-4886, E-ISSN 1090-2430, Vol. 169, no 2, p. 298-306Article in journal (Refereed)
    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.

  • 5.
    Jerregård, Helena
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Åkerud, Peter
    Division of Molecular Neurobiology, MBB, Karolinska Institute, Stockholm, Sweden.
    Arenas, Ernest
    Division of Molecular Neurobiology, MBB, Karolinska Institute, Stockholm, Sweden.
    Hildebrand, Claes
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Fibroblast-like cells from rat plantar skin and neurotrophin-transfected 3T3 fibroblasts influence neurite growth from rat sensory neurons in vitro2001In: Journal of Neurocytology, ISSN 0300-4864, E-ISSN 1573-7381, Vol. 29, no 9, p. 653-663Article in journal (Refereed)
    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.

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