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


OBJECTIVE
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.


METHODS
This is a randomized animal study.


METHODS
The SC-NSPCs were suspended in 100 μL 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 β-actin as the control group.


RESULTS
In vitro, SP (0.01-10 μmol/L) increased the proliferation of cultured SC-NSPCs, with a peak increase of 35±2% at the 0.1 μmol/L concentration. Substance P of 0.1 μmol/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 μM). Intrathecal infusion of SP (1 μmol/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 μmol/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 μmol/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 and p38 MAP kinases are important signaling proteins in this process.
Department of Neurosurgery, Kyungpook National University Hospital, 50 Samduk-2-ga, Jung-gu, Daegu 700-721, Republic of Korea. Electronic address: nskimkt7@gmail.com.
Kyoung-Tae
KT
Kim
Department of Neurosurgery, Kyungpook National University Hospital, 50 Samduk-2-ga, Jung-gu, Daegu 700-721, Republic of Korea.
Hye-Jeong
HJ
Kim
Department of Neurosurgery, Kyungpook National University Hospital, 50 Samduk-2-ga, Jung-gu, Daegu 700-721, Republic of Korea.
Dae-Chul
DC
Cho
Department of Physiology, School of Medicine, Kyungpook National University, 680 Gukchaebosang-ro, Jung-gu, Daegu 700-842, Republic of Korea.
Jae-Sung
JS
Bae
Department of Neurosurgery, College of Medicine, Chung-Ang University Hospital, 224-1 Heukseok dong, Dongjak-gu, Seoul 156-755, Republic of Korea.
Seung-Won
SW
Park
D000818:Animals; D049109:Cell Proliferation; D002478:Cells, Cultured; D020935:MAP Kinase Signaling System; D058953:Neural Stem Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology / Q000637:transplantation*; D051381:Rats; D013119:Spinal Cord Injuries / Q000628:therapy*; D033581:Stem Cell Transplantation; D013373:Substance P / Q000494:pharmacology*
D013373:Substance P
ERK; Mitogen-activated protein kinase signaling pathway; Neural stem/progenitor cell; Neuronal regeneration; Spinal cord injury; Substance P; p38