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, 29 (8), 1294-303

Bone Marrow Stromal Cells Produce Long-Term Pain Relief in Rat Models of Persistent Pain

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Bone Marrow Stromal Cells Produce Long-Term Pain Relief in Rat Models of Persistent Pain

Wei Guo et al. Stem Cells.

Abstract

Chronic pain conditions are difficult to treat and are major health problems. Bone marrow stromal cells (BMSCs) have generated considerable interest as a candidate for cell-based therapy. BMSCs are readily accessible and are easy to isolate and expand ex vivo. Clinical studies show that direct injection of BMSCs does not produce unwanted side effects and is well tolerated and safe. Here, we show that a single systemic (intravenous) or local injection (into the lesion site) of rat primary BMSCs reversed pain hypersensitivity in rats after injury and that the effect lasted until the conclusion of the study at 22 weeks. The pain hypersensitivity was rekindled by naloxone hydrochloride, an opioid receptor antagonist that acts peripherally and centrally, when tested at 1-5 weeks after BMSC infusion. In contrast, naloxone methiodide, a peripherally acting opioid receptor antagonist, only rekindled hyperalgesia in the first 3 weeks of BMSC treatment. Focal downregulation of brainstem mu opioid receptors by RNA interference (RNAi) reversed the effect of BMSCs, when RNAi was introduced at 5- but not 1-week after BMSC transplantation. Thus, BMSCs produced long-term relief of pain and this effect involved activation of peripheral and central opioid receptors in distinct time domains. The findings prompt studies to elucidate the cellular mechanisms of the BMSC-induced pain relieving effect and translate these observations into clinical settings.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

The authors indicate no potential conflicts of interest.

Figures

Figure 1
Figure 1
Characterization of bone marrow stromal cells (BMSCs) derived from the rat. (A): An example of BMSC culture at 5 days after plating. Note spindle-shaped cell bodies with a round nucleus and thin processes. Scale bar = 20 µm. (B, C): Flow cytometry analysis of cultured BMSCs at 5 days after plating. Left, SSC versus FSC scatter graphs; Right, plots of the number of positive immunoreactive cells versus relative fluorescence intensities. In the examples in (B, C), 90% of cells were positive for CD90 (B) and only 0.47% of cells were positive for CD45 (C). The CD90+ and CD45 surface marker phenotype is typical of BMSCs devoid of hematopoietic contamination. Isotype-matched IgG antibodies (IgG2a in (B) and IgG2b in (C)) were used as controls. Abbreviations: #Cells, number of positive immunoreactive cells; FITC, fluorescein isothiocyanate; FSC, forward scatter; SSC, side scatter.
Figure 2
Figure 2
Bone marrow stromal cell (BMSC) reversed pain hypersensitivity after tendon injury. (A): Cultured cells were infused through a tail vein at 0.2 ml with 1.5 × 103 (low-dose, MSC-low) or 1.5 × 106 (high-dose, MSC-high) BMSCs after 7 day-tendon ligation (TL). Control infusions were culture medium/phosphate-buffered saline, the supernatant of cell suspension (SuperN), and cells (1.5 × 106 BMSCs) after 20 passages (N-pass). When compared with low-dose BMSCs and controls, infusion of 1.5 × 106 BMSCs significantly increased EF50s from 1 day to 5 months after infusion. Asterisks denote significant differences versus 7 day-TL: *, p < .05; ***, p < .001. Cross signs denote significant differences versus medium: +, p < .05; ++, p < .01; +++, p < .001. (B): Infusion of BMSC reversed pain hypersensitivity after chronic constriction injury (CCI) of the infraorbital nerve of the rat. When compared with control (culture medium), infusion of BMSCs significantly increased EF50s from 1 day to 4 months after infusion. Asterisks denote significant differences versus 7 day-CCI: ***, p < .001. Cross signs denote significant differences versus medium: +++, p < .001. Error bars represent 95% confidence intervals of EF50s. Abbreviations: CCI, chronic constriction injury; PBS, phosphate-buffered saline; TL, tendon ligation.
Figure 3
Figure 3
Local injection of bone marrow stromal cells (BMSCs) at the site of injury reversed tendon injury-induced mechanical sensitivity. When compared with medium control, the local injection of BMSC reversed pain hypersensitivity starting from 1 day after cell injection. Note that the lower dose of BMSCs (0.02 ml, 1.5 × 105 cells) produced shorter lasting increase in EF50s when compared with the higher dose (0.05 ml, 3.75 × 105 cells). Asterisks denote significant differences versus 7 day-tendon ligation: ***, p < .001. Cross signs denote significant differences versus medium: +, p < .05; +++, p < .001. Error bars represent 95% confidence intervals of EF50s. Abbreviations: TL, tendon ligation.
Figure 4
Figure 4
Effect of intravenous bone marrow stromal cells (BMSCs) on early and late phases of mechanical hypersensitivity after tendon injury. BMSCs were infused at 3 days (A) or 2 and 4 months (B) after tendon ligation (TL; MSC-TL). Rats receiving sham operation (MSC-Sh) also was infused with BMSCs at 3 days after the surgery. There were significant increases in EF50s after BMSC infusion. Asterisks denote significant differences versus 3 day-TL/Sh or 2/4 months TL: *, p < .05; **, p < .01; ***, p < .001. Cross signs denote significant differences versus medium: +, p < .05; ++, p < .01; +++, p < .001. Error bars represent 95% confidence intervals of EF50s. Abbreviations: Sh, sham; TL, tendon ligation.
Figure 5
Figure 5
Effects of opioid receptor antagonists on bone marrow stromal cell-produced attenuation of mechanical hypersensitivity. A significant reduction of EF50 after naloxone (NX and NXm) indicates rekindling of mechanical hypersensitivity. Asterisks denote significant differences between naloxone and saline-treated rats: *, p < .05; **, p < .01; ***, p < .001. Error bars represent 95% confidence intervals of EF50s. Abbreviations: MSC, bone marrow stromal cell; NX, nalox-one hydrochloride; NXm, naloxone methiodide; TL, tendon ligation.
Figure 6
Figure 6
Downregulation of mu opioid receptors (MOR) in rostral ventromedial medulla (RVM) on bone marrow stromal cell (BMSCs) produced attenuation of mechanical hypersensitivity. (A): GFP staining illustrates transfer of shRNA into the RVM site. Green fluorescence was distributed in the region of nucleus raphe magnus (NRM), the major nucleus of the RVM. Scale bar = 250 µm. (B–G): Double immunofluorescence staining of RVM neurons in rats receiving scrambled control shRNA (B–D) or MOR-shRNA (E–G). RVM neurons exhibit intense GFP immunostaining (B, E; green), indicating transfer of shRNA into these neurons. GFP colocalizes (D; yellow) with MOR staining (red) (C, D), confirming transfer of shRNA into MOR-containing neurons. MOR-shRNA transfer led to reduction of MOR-like immunoreactivity (C, F). Scale bar = 25 µm. (H): Western immunoblot illustrating downregulation of MOR after transferring MOR-shRNA at 5 weeks after BMSC infusion. Beta-actin was used as a loading control. When compared with the control (5 days after Ctrl-shRNA transfer), the MOR levels were significantly reduced at 1–7 days (p < .01, n = 3–5) and returned to the control level at 10–21 days after MOR-shRNA transfer. Error bars represent SEM. (I, J): shRNAs were transferred into RVM at 5 weeks (I) and 1 week (J) after BMSC infusion. Significant reduction of EF50s was observed in rats receiving shRNA at 5 weeks but not 1 week after BMSC treatment. Asterisks denote significant differences versus 1/5 week-MSC: ***, p < .001. Cross signs denote significant differences versus Ctrl-shRNA: +, p < .05; +++, p < .001. Error bars represent 95% confidence intervals of EF50s. Abbreviations: ctrl, control; GFP, green fluorescent protein; MOR, mu opioid receptors; NRM, nucleus raphe magnus; Py, pyramidal tract; RPa, nucleus raphe pallidus; shRNA, small hairpin RNA; TL, tendon ligation.

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