Neurotropin inhibits both capsaicin-induced substance P release and nerve growth factor-induced neurite outgrowth in cultured rat dorsal root ganglion neurones.

BACKGROUND: Neurotropin (NTP), a biological extract from rabbit skin inoculated with vaccinia virus, is an effective analgesic and anti-allergic agent, and has antipruritic effects in various dermatoses including eczema, dermatitis and urticaria. In patients receiving haemodialysis who have pruritus, NTP appears to exert its antipruritic effect by lowering the plasma levels of substance P (SP), but its underlying mechanisms are not fully understood. AIM: To investigate the antipruritic mechanisms of NTP. METHODS: The effects of NTP on capsaicin-induced SP release from neonatal rat dorsal root ganglion (DRG) neurones were assessed by measuring SP concentrations in culture media by a competitive ELISA. The effects of NTP on nerve growth factor (NGF)-induced neurite outgrowth were assessed by measuring the length of the longest process of cultured DRG neurones. The neuronal cytotoxicity of NTP was determined using a methylthiazole tetrazolium cytotoxicity assay. RESULTS: NTP dose-dependently inhibited capsaicin-induced release of SP from cultured DRG neurones, whereas NTP alone had no effect on SP release. Moreover, NTP dose-dependently inhibited NGF-induced neurite outgrowth in cultured DRG neurones. NTP had no observable cytotoxicity. CONCLUSIONS: These results suggest that NTP exerts its antipruritic effects by inhibiting both SP release and neurite outgrowth of cutaneous sensory nerves.

In vitro model for penetration of sensory nerve fibres on a Matrigel basement membrane: implications for possible application to intractable pruritus.

BACKGROUND: Epidermal hyperinnervation occurs in dermatoses with intractable pruritus, such as atopic dermatitis, suggesting that the hyperinnervation is partly responsible for abnormal itch perception. OBJECTIVES: To investigate the mechanisms of penetration of sensory nerve fibres into the basement membrane of the skin. METHODS: A rat dorsal root ganglion neurone culture system consisting of Matrigel and a Boyden chamber containing a nerve growth factor (NGF) concentration gradient was used. In some experiments, matrix metalloproteinase (MMP) blockers and semaphorin 3A (Sema3A) were added to the culture system. Matrigel-coated membranes were stained with anti-Tau antibody, and the number of nerve fibres that crossed the membrane was counted. Expression of MMPs in the cultured neurones was examined at mRNA and protein levels by quantitative reverse transcription-polymerase chain reaction and immunocytochemistry, respectively. The activity was also examined by zymography. RESULTS: Nerve fibres penetrated into Matrigel in the presence of an NGF concentration gradient, which was dose-dependently inhibited by GM6001, a broad-spectrum MMP inhibitor. Transcripts for MMP2, but not MMP9, were increased in the cultured neurones, and the penetration was dose-dependently inhibited by MMP-2 blockers. MMP-2 and its activity were partially localized on the NGF-responsive growth cones. NGF also upregulated pro-MMP-2 activation molecules in the cultured neurones. Sema3A stimulation showed the opposite effects on these NGF-dependent events. Interestingly, MMP2 expression was modulated by extracellular matrix (ECM) substrates for this enzyme. CONCLUSIONS: Membrane-associated MMP-2 contributes to penetration of nerve fibres into Matrigel through modulation by axonal guidance molecules and/or ECM. These findings provide insight for understanding the development of intractable pruritus involving epidermal nerve density.