Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 5, 79
eCollection

Long-lasting Effects of Human Mesenchymal Stem Cell Systemic Administration on Pain-Like Behaviors, Cellular, and Biomolecular Modifications in Neuropathic Mice

Affiliations

Long-lasting Effects of Human Mesenchymal Stem Cell Systemic Administration on Pain-Like Behaviors, Cellular, and Biomolecular Modifications in Neuropathic Mice

Dario Siniscalco et al. Front Integr Neurosci.

Abstract

Background: Neuropathic pain (NP) is an incurable disease caused by a primary lesion in the nervous system. NP is a progressive nervous system disease that results from poorly defined neurophysiological and neurochemical changes. Its treatment is very difficult. Current available therapeutic drugs have a generalized nature, sometime acting only on the temporal pain properties rather than targeting the several mechanisms underlying the generation and propagation of pain.

Methods: Using biomolecular and immunohistochemical methods, we investigated the effect of the systemic injection of human mesenchymal stem cells (hMSCs) on NP relief. We used the spared nerve injury (SNI) model of NP in the mouse. hMSCs were injected into the tail vein of the mouse. Stem cell injection was performed 4 days after sciatic nerve surgery. Neuropathic mice were monitored every 10 days starting from day 11 until 90 days after surgery.

Results: hMSCs were able to reduce pain-like behaviors, such as mechanical allodynia and thermal hyperalgesia, once injected into the tail vein. An anti-nociceptive effect was detectable from day 11 post surgery (7 days post cell injection). hMSCs were mainly able to home in the spinal cord and pre-frontal cortex of neuropathic mice. Injected hMSCs reduced the protein levels of the mouse pro-inflammatory interleukin IL-1β and IL-17 and increased protein levels of the mouse anti-inflammatory interleukin IL-10, and the marker of alternatively activated macrophages CD106 in the spinal cord of SNI mice.

Conclusion: As a potential mechanism of action of hMSCs in reducing pain, we suggest that they could exert their beneficial action through a restorative mechanism involving: (i) a cell-to-cell contact activation mechanism, through which spinal cord homed hMSCs are responsible for switching pro-inflammatory macrophages to anti-inflammatory macrophages; (ii) secretion of a broad spectrum of molecules to communicate with other cell types. This study could provide novel findings in MSC pre-clinical biology and their therapeutic potential in regenerative medicine.

Keywords: cell transplantation; human mesenchymal stem cells; macrophages; neuropathic pain.

Figures

Figure 1
Figure 1
(A) Effects of human mesenchymal stem cells on reflex withdrawal responses (g, mean ± SEM) to mechanical noxious stimuli in SNI mice. The onset of SNI-induced mechanical allodynia was evaluated in ipsilateral sides every 10 days starting from day 11 until 90 days after surgery. Mice showed a significant reduction in the threshold to mechanical stimulation in the ipsilateral paw (*p < 0.05 vs. sham-operated mice) after SNI surgery. Human MSC treatment (on day 4 after SNI surgery, as indicated by the arrow) prevented the appearance of mechanical allodynia at 11, 20, 30, 40, 50, 60, 70, 80, and 90 days post-SNI (°p < 0.05 vs. SNI mice). (B) Effects of human mesenchymal stem cells on reflex withdrawal responses (s, mean ± SEM) to thermal noxious stimuli in SNI mice. The onset of SNI-induced thermal hyperalgesia was evaluated in ipsilateral sides every 10 days starting from day 11 until 90 days after surgery. SNI mice showed a significant reduction in withdrawal latency to radiant heat in the ipsilateral paw (*p < 0.05 vs. sham-operated mice). Human MSC treatment (on day 4 after SNI surgery, as indicated by the arrow) prevented the appearance of thermal hyperalgesia 11, 20, 30, 40, 50, 60, 70, 80, and 90 days post-SNI (°p < 0.05 vs. SNI mice). (C) Rotarod motor testing results. The effects of human mesenchymal stem cell injection on motor performance in the Rotarod test is shown. Systemic administration of hMSCs in tail vein of SNI mice had no effect on motor function compared with vehicle treated SNI mice. Human MSCs were injected on day 4 after the SNI surgery. Results are expressed as the mean ± SEM of the latency (s; n = 5 mice/group). *p < 0.05 vs. sham/vehicle.
Figure 2
Figure 2
(A) Representative agarose gel blot analysis for human GAPDH gene of 30 days-SNI/hMSCs mice following RT-PCR is shown. Lane (1) lung; lane (2) sciatic nerve; lane (3) ventral L4–L5 spinal cord and (4) dorsal L4–L5 spinal cord; lane (5) pre-frontal cortex; lane (6) DNA ladder marker. The analysis of mRNA levels was carried out by the “Gel Doc 2000 UV System” (Bio-Rad, Hercules, CA, USA). RT-PCR analysis revealed a preferential accumulation of hMSCs to the L4–L5 spinal cord (both ventral and dorsal areas) and pre-frontal cortex. Very weak band was found from lung. No GAPDH band was detectable in sciatic nerve, thus revealing the specificity of hMSC engraftment at the NP controlling and processing areas. (B) Representative cross-section of mouse whole L4–L5 spinal cord area from hMSC-treated 30 days-neuropathic mice (26 days post-hMSC injection). (B’) Representative cross-section of mouse whole L4–L5 spinal cord area from vehicle treated 30 days-neuropathic mice showing no staining for human specific CD73. (C,D) Human MSCs expressed lineage-specific antigens at 26 days post-injection in vivo. Representative fluorescent photomicrograph of hMSCs showing immunocytochemistry for CD73. Area in white rectangle insets is shown: (C) ventral spinal cord; (D) dorsal spinal cord; left: cell nuclei were counterstained with DAPI (blue fluorescence); middle: CD73-positive hMSCs emitted red fluorescence; right: pictures with both red and blue fluorescence were merged. Scale bars: 100 μm.
Figure 3
Figure 3
Representative western blot analysis for IL-1β, IL-17, IL-10, CD206, and housekeeping β-tubulin mouse proteins are shown. The semi-quantitative analysis of protein levels was carried out by the “Gel Doc 2000 UV System” (Bio-Rad, Hercules, CA, USA). Human MSC treatment (on day 4 after SNI surgery) reduced the protein levels of pro-inflammatory interleukins IL-1β and IL-17 in hMSC-treated 30 days-neuropathic mice (SNI + hMSCs) respect to vehicle treated 30 days-neuropathic mice (SNI + vehicle). Human MSC treatment (on day 4 after SNI surgery) increased the protein levels of anti-inflammatory interleukin IL-10 and activated anti-inflammatory macrophage marker CD206 in hMSC-treated 30 days-neuropathic mice (SNI + hMSCs) respect to vehicle treated 30 days-neuropathic mice (SNI + vehicle). Upper graph shows IL-1β/β-tubulin normalized values as obtained by immunoblot analysis. Human MSC treatment (on day 4 after SNI surgery) reduced the protein levels of IL-1β interleukin in hMSC-treated 30 days-neuropathic mice (SNI + hMSCs) respect to vehicle treated 30 days-neuropathic mice (SNI + vehicle). ANOVA, followed by Student–Neuman–Keuls post hoc test, was used to determine the statistical significance among groups. *p < 0.01 was considered statistically significant. Lower graph shows CD206/β-tubulin normalized values as obtained by immunoblot analysis. Human MSC treatment (on day 4 after SNI surgery) increased the protein levels of activated anti-inflammatory macrophage marker CD206 in hMSC-treated 30 days-neuropathic mice (SNI + hMSCs) respect to vehicle treated 30 days-neuropathic mice (SNI + vehicle). ANOVA, followed by Student–Neuman–Keuls post hoc test, was used to determine the statistical significance among groups. *p < 0.01 was considered statistically significant.
Figure 4
Figure 4
Representative cross-section of mouse L4–L5 spinal cord from 30 days-neuropathic mice. (A) First: IL-17 positive profiles (green fluorescent) in vehicle treated 30 days-neuropathic mice (SNI + vehicle). Second: CD4 positive profiles (red fluorescent) in vehicle treated 30 days-neuropathic mice. Third: double labeling of IL-17 and CD4 positive profiles. Fourth: higher magnification showing that the cells expressing IL-17 were T lymphocytes. Cell nuclei were counterstained with DAPI (blue fluorescence). Scale bars: 100 μm. (B) First: IL-1β positive profiles (green fluorescent) in vehicle treated 30 days-neuropathic mice. Second: GFAP positive profiles (red fluorescent) in vehicle treated 30 days-neuropathic mice. Third: double labeling of GFAP and IL-1β positive profiles. Fourth: higher magnification indicating that the cells expressing IL-1β were astrocytes. Cell nuclei were counterstained with DAPI (blue fluorescence). Scale bars: 100 μm. (C) First: IL-1β profiles (green fluorescent) in hMSC-treated 30 days-neuropathic mice (SNI + hMSCs). Second: IL-17 profiles (green fluorescent) in hMSC-treated 30 days-neuropathic mice (SNI + hMSCs). The absence of positive staining indicates that injected hMSCs were able to reduced the protein levels of pro-inflammatory interleukins IL-1β and IL-17, confirming western blot analysis results.

Similar articles

See all similar articles

Cited by 36 PubMed Central articles

See all "Cited by" articles

References

    1. Ankrum J., Karp J. M. (2010). Mesenchymal stem cell therapy: two steps forward, one step back. Trends. Mol. Med. 16, 203–20910.1016/j.molmed.2010.02.005 - DOI - PMC - PubMed
    1. Beggs K. J., Lyubimov A., Borneman J. N., Bartholomew A., Moseley A., Dodds R., Archambault M. P., Smith A. K., McIntosh K. R. (2006). Immunologic consequences of multiple, high-dose administration of allogeneic mesenchymal stem cells to baboons. Cell Transplant. 15, 711–72110.3727/000000006783981503 - DOI - PubMed
    1. Beyth S., Borovsky Z., Mevorach D., Liebergall M., Gazit Z., Aslan H., Galun E., Rachmilewitz J. (2005). Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T-cell unresponsiveness. Blood 105, 2214–221910.1182/blood-2004-07-2921 - DOI - PubMed
    1. Bonfield T. L., Caplan A. I. (2010). Adult mesenchymal stem cells: an innovative therapeutic for lung diseases. Discov. Med. 9, 337–345 - PubMed
    1. Bourquin A. F., Süveges M., Pertin M., Gilliard N., Sardy S., Davison A. C., Spahn D. R., Decosterd I. (2006). Assessment and analysis of mechanical allodynia-like behavior induced by spared nerve injury (SNI) in the mouse. Pain 122, 14.e1–14.e1410.1016/j.pain.2005.10.036 - DOI - PubMed

LinkOut - more resources

Feedback