Korean J Physiol Pharmacol.  2017 Mar;21(2):153-160. 10.4196/kjpp.2017.21.2.153.

Effect of subcutaneous treatment with human umbilical cord blood-derived multipotent stem cells on peripheral neuropathic pain in rats

• Lee MJ
• Yoon TG
• Kang M
• Kim HJ
• Kang KS

• KMID: 2371033
• DOI: http://doi.org/10.4196/kjpp.2017.21.2.153

Abstract

In this study, we aim to determine the in vivo effect of human umbilical cord blood-derived multipotent stem cells (hUCB-MSCs) on neuropathic pain, using three, principal peripheral neuropathic pain models. Four weeks after hUCB-MSC transplantation, we observed significant antinociceptive effect in hUCB-MSC-transplanted rats compared to that in the vehicle-treated control. Spinal cord cells positive for c-fos, CGRP, p-ERK, p-p 38, MMP-9 and MMP 2 were significantly decreased in only CCI model of hUCB-MSCs-grafted rats, while spinal cord cells positive for CGRP, p-ERK and MMP-2 significantly decreased in SNL model of hUCB-MSCs-grafted rats and spinal cord cells positive for CGRP and MMP-2 significantly decreased in SNI model of hUCB-MSCs-grafted rats, compared to the control 4 weeks or 8weeks after transplantation (p<0.05). However, cells positive for TIMP-2, an endogenous tissue inhibitor of MMP-2, were significantly increased in SNL and SNI models of hUCB-MSCs-grafted rats. Taken together, subcutaneous injection of hUCB-MSCs may have an antinociceptive effect via modulation of pain signaling during pain signal processing within the nervous system, especially for CCI model. Thus, subcutaneous administration of hUCB-MSCs might be beneficial for improving those patients suffering from neuropathic pain by decreasing neuropathic pain activation factors, while increasing neuropathic pain inhibition factor.

Keyword

Cord blood stem cell transplantation; Neuropathic pain; Rat subcutaneous injection; Tissue inhibitor of metalloproteinase 2

MeSH Terms

Animals
Cord Blood Stem Cell Transplantation
Humans*
Injections, Subcutaneous
Multipotent Stem Cells*
Nervous System
Neuralgia*
Rats*
Spinal Cord
Tissue Inhibitor of Metalloproteinase-2
Umbilical Cord*
Tissue Inhibitor of Metalloproteinase-2

Figure

• 

  Fig. 1 The effect of hUCB-MSCs on mechanical hypersensitivity in the three neuropathic pain models.After establishment of neuropathic pain behavior (14 days after CCI, SNL, and SNI), PBS (n=6∼7) or hUCB-MSCs (n=10∼12, 1×105 cells/50 µl in PBS) were subcutaneously injected into the plantar. “0 day drug injection start point” of arrows stands for transplantation of hUCB-MSCs. Mechanical hypersensitivity measured by withdrawal threshold was assessed after 1 and 2 days, and every week for a month thereafter. In the three neuropathic pain models, mechanical threshold of the hUCB-MSCs group was higher than control group. BL indicates a withdrawal threshold at base line, which is the start point of neuropathic pain animal model establishment. The data are presented as mean±S.E.M. *p<0.05, ***p<0.001 indicate a significant difference compared to control group.

• 

  Fig. 2 Immunohistochemical analysis using the pain markers in the laminae I-II layers of the ipsilateral L4∼L5 spinal dorsal horns in the three neuropathic pain models.The laminae I∼II layers of the ipsilateral L4∼L5 spinal dorsal horns in CCI and SNL models were dissected and sampled on 4 weeks, while those layers in SNI models dissected on 8 weeks after transplantation of hUCB-MSCs. The number of cells positive for pain-related antibodies such as c-fos, CGRP, p-ERK and p-p38 was decreased in the hUCB-MSCs group (n=11 in CCI, n=10 in SNL, n=12 in SNI) compared to control group (n=6 in CCI, n=7 in SNL, n=6 in SNI). Positive cells area (%) presents the mean percentages of c-fos or CGRP or p-ERK or p-p38-immunoreactive (IR) neurons relative to the total number of neurons in the laminae I∼II layers of the ipsilateral L4∼L5 spinal dorsal horns. Statistical significance of difference was determined by Duncan's t-test. A p<0.05 was regarded as statistically significant. The data are presented as mean±S.E.M. *p<0.05, **p<0.01 and ***p<0.001 indicate a significant difference compared to control group. All of the micro-graphics are from the CCI group. A, c-fos; B, CGRP; C, p-ERK; D, p-p38. Scale bars, 200 µm

• 

  Fig. 3 Immunohistochemistry and western blot analysis for MMP-9, MMP-2, and TIMP-2 in the spinal cord.The laminae I-II layers of the ipsilateral L4-L5 spinal dorsal horns and L4-L5 spinal cord in CCI and SNL models were dissected and sampled on 4 weeks, while those layers and that spinal cord in SNI models dissected on 8 weeks after transplantation of hUCB-MSCs. The cells positive for MMP-9, MMP-2, and TIMP-2 in the laminae I-II layers of the ipsilateral L4-L5 spinal dorsal horn were analyzed in the three neuropathic pain models. Positive cells area (%) presents the mean percentages of MMP-9 or MMP-2 or TIMP-2 immunoreactive (IR) neurons relative to the total number of neurons in the laminae I-II layers of the ipsilateral L4-L5 spinal dorsal horns. Expression of MMP-9 was only reduced in CCI model. In all of the neuropathic pain models, expression of MMP-2 was significantly decreased in the hUCB-MSCs group (n=11 in CCI, n=10 in SNL, n=12 in SNI) compared to control group (n=6 in CCI, n=7 in SNL, n=6 in SNI). In the hUCB-MSCs group, TIMP-2-positive cells were significantly increased in SNL and SNI models. Results for expression of those antibodies was similar in the western blot analysis. Statistical significance of difference was determined by Duncan's t-test. A p<0.05 was regarded as statistically significant. The data are presented as mean±S.E.M. *p<0.05, **p<0.01 indicate a significant difference compared to control group. The micrographs A and B are taken from the CCI group and C from the SNL group. A: MMP-9, B: MMP-2, C: TIMP-2, D: western blot analysis. Scale bars, 200 µm.

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