Khan, Michael M. T.; (1996) Neuropeptides and cytokines in regenerating and degenerating peripheral nerve. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis concerns the role of neuropeptides and cytokines in regeneration of injured peripheral nerve. A novel experimental technique is described for the rapid preparation of pure cell populations from rat sciatic nerve, involving immunoselection by "panning". Forskolin elevates Schwann cell cAMPi in vitro, permitting growth factor-induced proliferation and expression of nerve growth factor (NGF) and of the transcription factor suppressed cAMP inducible POU (SCIP). The neuropeptide calcitonin gene-related peptide (CGRP) acts through cAMPi in other systems and is upregulated in regenerating motor axons following peripheral nerve injury. I show that CGRP and isoprenaline elevate cAMPi acutely in non-neuronal cells derived from rat sciatic nerve, but that this cAMPi elevation rapidly attenuates due, probably, to the desensitisation of receptors by cAMP-dependant protein kinase (PKA) and the (β-adrenoceptor kinase (βARK), respectively. CGRP fails to promote Schwann cell proliferation, in vitro, due to the time course of desensitisation rather than lack of signalling downstream of cAMPi, since CGRP elevates Schwann cell SCIP, and inhibits fibroblast proliferation. Degradation of CGRP by Schwann cell endopeptidase 24.11. may reduce desensitisation in vivo. Supraphysiological magnesium (Mg++) concentrations enhance receptor / G protein coupling, allowing maintenance of cAMPi elevation in response to CGRP. Consequently, previously desensitising levels of CGRP and isoprenaline are now mitogenic and increase NGF synthesis. In the second section, I show that interleukin-1β (IL-lβ) promotes Schwann cell proliferation and survival in culture and, with tumour necrosis factor a (TNFa), additively increases NGF production. These responses are lost with time and thus are not apparent in cells purified by conventional methods. IL-1β is active alone and stimulates several distinct intracellular signals: these probably include PKA, a pertussis toxin sensitive G protein, prostaglandin E2 (PGE2) and a tyrosine kinase or protein kinase C (PKC). Inhibiting any of these transduction signals reduces IL-1β action. Gamma-interferon (γ-IFN) dramatically enhances IL-lβ stimulated NGF synthesis, yet inhibits proliferation. When PGE2 synthesis or PKA are downregulated, IL-1β becomes weakly mitogenic for fibroblasts but no longer promotes NGF synthesis. Transforming growth factor type β (TGFβ), a macrophage product, does not influence quiescent Schwann cells, but inhibits mitogen induced proliferation. A model is proposed whereby the responses of non-neuronal cells after axotomy are regulated by a combination of axon and macrophage-derived signals, with a priming action by T cells. Clinical ramifications of the work are then discussed.

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