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.
Linköping: Linköpings universitet , 2001. , 92 p.
2001-11-02, Berzeliussalen, Universitetssjukhuset, Linköping, 09:00 (Swedish)