Various neurotrophic factors that promote axonal regeneration have been investigated in vivo, but the signals that prompt neurons to send out processes in peripheral nerves after axotomy are not well understood. Previously, we have shown oxidized galectin-1 (GAL-1/Ox) promotes initial axonal growth after axotomy in peripheral nerves. However, the mechanism by which GAL-1/Ox promotes axonal regeneration remains unclear and is the subject of the present study. To identify possible target cells of GAL-1/Ox, a fluorescently labeled recombinant human GAL-1/Ox (rhGAL-1/Ox) was incubated with DRG neurons, Schwann cells, and intraperitoneal macrophages from adult rats. Only the cell surfaces of intraperitoneal macrophages bound the rhGAL-1/Ox, suggesting that these cells possess a receptor for GAL-1/Ox. Experiments examining tyrosine phosphorylation revealed that rhGAL-1/Ox stimulated changes in signal transduction pathways in these macrophages. These changes caused macrophages to secrete an axonal growth-promoting factor. This was demonstrated when conditioned media of macrophages stimulated with rhGAL-1/Ox in 48 hr culture strongly enhanced axonal regeneration from transected-nerve sites of DRG explants. Furthermore, activated macrophage-conditioned media also improved Schwann cell migration from the transected-nerve sites. From these results, we propose that axonal regeneration occurs in axotomized peripheral nerves as a result of cytosolic reduced galectin-1 being released from Schwann cells and injured axons, which then becomes oxidized in the extracellular space. Oxidized galectin-1 then stimulates macrophages to secrete a factor that promotes axonal growth and Schwann cell migration, thus enhancing peripheral nerve regeneration.
Interleukin-6 (IL-6) is a neurotrophic cytokine, however, its direct effect on nerve regeneration has not been well characterized. We therefore examined the effect of IL-6 on neurite regeneration using the rat dorsal root ganglion. IL-6 significantly enhanced neurite regeneration from transected nerve terminals. We also examined the mRNA expression of IL-6 family cytokines and their receptors during the regeneration. The mRNA expressions of IL-6, IL-6 receptor, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF) receptor alpha, and LIF receptor beta showed no significant differences by the addition of IL-6. In contrast, IL-6 enhanced the mRNA expression of gp130 and CNTF. In addition, CNTF significantly increased neurite regeneration when added exogenously. Our data suggest that IL-6 enhanced regeneration via up-regulating CNTF expression.
1. Our previous study demonstrated that cultured macrophages release neurotrophic factors spontaneously. In a histological study of Wallerian degeneration, macrophages phagocytosed myelin debris and expressed activated markers. 2. To investigate the role of myelin-stimulated macrophages on neurite regeneration, we prepared conditioned media from cultured mouse peritoneal macrophages which had phagocytosed a myelin fraction. This conditioned media enhanced both neurone survival and neurite regeneration of adult dorsal root ganglia (DRG) neurons compare to conditioned media from macrophage cultures without myelin. 3. The production of the neurotrophic supernatant was dose-dependent on myelin fraction and specific for myelin because supernatants from macrophages incubated with LPS (lipoplysaccharide), MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine) or latex beads were not neurotrophic. 4. The neurotrophic factors from myelin-stimulated macrophages were different from spontaneously released macrophage factors as they differed in heat-sensitivity. 5. These results suggest that myelin-stimulated macrophages contribute to axon regeneration after Wallerian degeneration.
A technique for isolation of adult Schwann cells (ScC) from dorsal root ganglia (DRG) is described. Decapsulated DRG explants embedded into type I collagen gels were cultured for 3 days in serum-free medium during which ScC migrated from the explant. These explants were then grown in serum-supplemented medium to allow ScC proliferation. On day 10 the number of ScC isolated from DRG explants per mouse was about 2.5 x 10(5), and the purity was greater than 95%. This culture system provided sufficient numbers of highly purified adult ScC in a shorter culture period (2-3 times) than other methods. We used ScC from this method to determine the age-related changes in attachment, growth, and survival of ScC cultured in serum-free medium. The attachment capacity of adult ScC on type I collagen or polylysine was similar to that of newborn ScC. However, the collagen promoted growth and survival of adult ScC but not that of neonatal ScC, indicating age-related differences of ScC properties in vitro.
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