Substance P stimulates proliferation of spinal neural stem cells in spinal cord injury via the mitogen-activated protein kinase signaling pathway

Abstract

BACKGROUND CONTEXT: Substance P (SP) is a neuropeptide that can influence neural stem/progenitor cell (NSPC) proliferation and neurogenesis in the brain. However, we could not find any experimental study that investigates SP action in the spinal cord. PURPOSE: The aims of our study were to investigate the potential of the neuropeptide SP in promoting the proliferation of spinal cord-derived NSPCs (SC-NSPCs) after spinal cord injury (SCI) and to clarify the roles of the mitogen-activated protein (MAP) kinase signaling pathway in the process. STUDY DESIGN: This is a randomized animal study. METHODS: The SC-NSPCs were suspended in 100 mu L of a neurobasal medium containing SP (binds neurokinin-1 receptor [NK1R]) or L-703,606 (NK1R antagonist) and cultured in a 96-well plate for 5 days. A cell proliferation assay was performed using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. A cord clipping method was used for the SCI model. Substance P and the NK1R antagonist (L-703,606) were infused intrathecally in SCI and sham models. Neural stem/progenitor cell proliferation was evaluated with immunostaining for bromodeoxyuridine (BrdU) and the immature neural marker nestin. An immunoblotting method was used for evaluating the MAP kinase signaling protein that contains extracellular signal-regulated kinases (ERKs and p38) and b-actin as the control group. RESULTS: In vitro, SP (0.01-10 mu mol/L) increased the proliferation of cultured SC-NSPCs, with a peak increase of 35 +/- 2% at the 0.1 mu mol/L concentration. Substance P of 0.1 mu mol/L continuously increased SC-NSPC proliferation from 6 hours to 5 days, whereas the proliferation decreased from 18% to 98% with L-703,606 (1-10 mu M). Intrathecal infusion of SP (1 mmol/L) for 7 days significantly increased the number of proliferating NPSCs (cells positive for both BrdU and nestin) in the spinal cord (by 120 +/- 17%, p<.05) in adult rats, but infusion of L-703,606 (10 mmol/L) significantly decreased the post-SCI induction of NPSC proliferation in the spinal cord (by 87 +/- 4%). Also, SP stimulates proliferation of SC-NSPCs via the MAP kinase signaling pathway, especially the phosphorylated ERK and phosphorylated p38 proteins. The phosphorylated ERK and phosphorylated p38 protein levels increased with SP (0.1 mu mol/L, p<.05). CONCLUSIONS: These data indicate that SP can promote proliferation of SC-NSPCs in SCI and normal conditions and have important roles in neuronal regeneration after SCI. Also, ERKs