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, 125 (8), 3226-40

Intrathecal Bone Marrow Stromal Cells Inhibit Neuropathic Pain via TGF-β Secretion

Intrathecal Bone Marrow Stromal Cells Inhibit Neuropathic Pain via TGF-β Secretion

Gang Chen et al. J Clin Invest.

Abstract

Neuropathic pain remains a pressing clinical problem. Here, we demonstrate that a local, intrathecal (i.t.) injection of bone marrow stromal cells (BMSCs) following lumbar puncture alleviates early- and late-phase neuropathic pain symptoms, such as allodynia and hyperalgesia, for several weeks in murine chronic constriction injury (CCI) and spared nerve injury models. Moreover, i.t. BMSCs reduced CCI-induced spontaneous pain and axonal injury of dorsal root ganglion (DRG) neurons and inhibited CCI-evoked neuroinflammation in DRGs and spinal cord tissues. BMSCs secreted TGF-β1 into the cerebrospinal fluid, and neutralization of TGF-β1, but not IL-10, reversed the analgesic effect of BMSCs. Conversely, i.t. administration of TGF-β1 potently inhibited neuropathic pain. TGF-β1 acted as a powerful neuromodulator and rapidly (within minutes) suppressed CCI-evoked spinal synaptic plasticity and DRG neuronal hyperexcitability via TGF-β receptor 1-mediated noncanonical signaling. Finally, nerve injury upregulated CXCL12 in lumbar L4-L6 DRGs, and this upregulation caused migration of i.t.-injected BMSCs to DRGs through the CXCL12 receptor CXCR4, which was expressed on BMSCs. BMSCs that migrated from the injection site survived at the border of DRGs for more than 2 months. Our findings support a paracrine mechanism by which i.t. BMSCs target CXCL12-producing DRGs to elicit neuroprotection and sustained neuropathic pain relief via TGF-β1 secretion.

Figures

Figure 10
Figure 10. Prolonged inhibition of SNI-induced mechanical allodynia by i.t. BMSCs in the early or late phase and its reversal by TGF-β1 Abs.
(A and B) Long-term inhibition of mechanical allodynia by early treatment with BMSCs (2.5 × 105 cells) via i.t. injection, given 4 days after SNI. Mechanical allodynia was tested using the paw withdrawal threshold (A) and by the frequency of response (percentage of response) to a single 0.16 gf filament (10 times) (B). *P < 0.05, compared with vehicle (PBS); n = 6 mice/group. (C and D) Sustained inhibition of mechanical allodynia by late treatment with i.t. BMSCs (2.5 × 105 cells), given 21 days after SNI. *P < 0.05, compared with vehicle; n = 6 mice/group. (E) Reversal of BMSC-induced inhibition of mechanical allodynia by i.t. TGF-β1–neutralizing Abs (4 μg), given 3 weeks after SNI. Arrows in AE indicate the time of injection. *P < 0.05, compared with the IgG control group; n = 5 mice/group. All data are expressed as the mean ± SEM. Statistical significance was determined by 2-way ANOVA, followed by Bonferroni’s post-hoc test.
Figure 9
Figure 9. Long-term survival of CM-Dil–labeled BMSCs in ipsilateral L5 DRGs following i.t. injection in CCI mice.
(A) Localization of CM-Dil–labeled BMSCs in ipsilateral L5 DRGs 3–56 days after i.t. injection. Scale bars: 50 μm (top row) and 10 μm (bottom row). Bottom panels are enlarged images of the top panels. Note that BMSCs were mainly localized at DRG borders. (B) Number of CM-Dil–labeled BMSCs in L5 DRGs 3–84 days after i.t. BMSC injection, given 4 days after CCI. n = 4 mice/group. (C) CM-Dil–labeled BMSCs expressed the stem cell marker CD90 in L5 DRGs 28 days after BMSC injection. Bottom panels are high-magnification merged images of the top panels. Scale bars: 50 μm (upper) and 10 μm (lower).
Figure 8
Figure 8. CXCL12/CXCR4 axis controls BMSC migration to lumbar DRGs and mediates the antiallodynic effect of BMSCs in CCI mice.
(A) Selective targeting of i.t.-injected BMSCs (CM-Dil labeled) to ipsilateral L4–L6 DRGs 3 days after i.t. injection (day 7 after CCI). Scale bar: 100 μm. Note that there was only limited migration of BMSCs to contralateral DRGs. (B) ELISA analysis showing CXCL12 expression in contralateral and ipsilateral L4–L6 DRGs on days 4 and 14 after CCI. *P < 0.05; n = 4 mice/group. (C) Chemotaxis (Transwell invasion) assay showing the migration of BMSCs in response to CXCL12 (0–100 ng/ml) and the inhibitory effect of the CXCR4 antagonist AMD3100 (5 mg/ml, 30 min). *P < 0.05, compared with the control group (no treatment); #P < 0.05; n = 4 wells from separate cultures. (D) Reduction of Cxcr4 mRNA levels in BMSCs treated with Cxcr4 siRNA (1 μg/ml for 18 h). *P < 0.05; n = 3 separate cultures. (E) Antiallodynic effect of i.t. BMSCs (2.5 × 105 cells) was compromised by pretreatment of BMSCs with Cxcr4 siRNA, but not with nontargeting control siRNA. Arrow indicates BMSC injection on day 14 after CCI. *P < 0.05, compared with the vehicle group; #P < 0.05; n = 5 mice/group. (F) Migration of CM-Dil–labeled BMSCs to ipsilateral L5 DRGs 7 days after i.t. injection (day 21 after CCI). Note that this migration was blocked by Cxcr4 siRNA. Scale bar: 50 μm. (G) Number of CM-Dil–labeled BMSCs in ipsilateral L4–L6 DRGs after the treatment shown in F. *P < 0.05; n = 5 mice/group. Statistical significance was determined by 1-way ANOVA, followed by Bonferroni’s post-hoc test (B and C), 2-way, repeated-measures ANOVA, followed by Bonferroni’s post-hoc test (E), or Student’s t test (D and G). All data are expressed as the mean ± SEM.
Figure 7
Figure 7. Exogenous TGF-β1 blocks CCI-induced increases in action potential frequency in whole-mount DRGs via the TGF-β1R.
(A) Traces of evoked action potentials in small-sized neurons of whole-mount DRGs from sham and CCI mice. (B) Frequency of action potentials and effects of CCI, TGF-β1, and SB431542. Note that the CCI-induced increase in action potential frequency was suppressed by TGF-β1 (2 or 10 ng/ml), and this suppression was abrogated by SB431542. *P < 0.05; #P < 0.05, compared with the CCI control. Statistical significance was determined by 1-way ANOVA, followed by Bonferroni’s post-hoc test; n = 5 neurons/group. All data are expressed as the mean ± SEM.
Figure 6
Figure 6. Exogenous TGF-β1 rapidly suppresses CCI-induced enhancement of excitatory synaptic transmission in lamina IIo neurons of spinal cord slices via the TGF-β1R.
(A) Traces showing sEPSCs in lamina IIo neurons of spinal cord slices. (B) Frequency and amplitude of sEPSCs. CCI (day 4) induced profound increases in sEPSC frequency and amplitude, which were suppressed by TGF-β1 (2 or 10 ng/ml). Note that TGF-β1 had no effect on the frequency or amplitude of sEPSCs in sham control spinal cord. *P < 0.05, compared with sham surgery; #P < 0.05 compared with the control group; n = 5 neurons/group. (C) The TGF-β1R antagonist SB431542 blocked TGF-β1–induced inhibition of sEPSC frequency and amplitude. *P < 0.05; n = 5 neurons/group. Statistical significance was determined by 1-way ANOVA, followed by Bonferroni’s post-hoc test. All data are expressed as the mean ± SEM.
Figure 5
Figure 5. BMSCs release TGF-β1 to inhibit neuropathic pain in CCI mice.
(A) ELISA analysis showing TGF-β1 and IL-10 release in BMSC culture medium and the effects of TNF (10 ng/ml, 60 min) and LPS (100 ng/ml, 60 min) on the release. *P < 0.05, compared with the respective control group; n = 8 separate cultures from different mice. (B) ELISA analysis showing increased TGF-β1 release in CSF 8 days after CCI and 4 days after i.t. delivery of 2.5 × 105 BMSCs. *P < 0.05, compared with naive and vehicle-treated cells; #P < 0.05; n = 4 mice/group. (C) Reversal of BMSC-induced inhibition of mechanical allodynia by TGF-β1–neutralizing Abs (4 μg, i.t.), but not by IL-10–neutralizing Abs (4 μg, i.t.) or control IgG (4 μg, i.t.). *P < 0.05, compared with the control IgG group; n = 5 mice/group. (D) Reduction of TGF-β1 release and expression in BMSCs following Tgfb1 siRNA treatment (1 μg/ml for 18 h). Both baseline release and evoked release by TNF (10 ng/ml, 1 h) were measured. *P < 0.05, compared with nontargeting siRNA; #P < 0.05; n = 4 separate cultures from different mice. (E) Antiallodynic effect of BMSCs (2.5 × 105 cells) was compromised by pretreatment of BMSCs with Tgfb1 siRNA (1 μg/ml for 18 h), but not with nontargeting control siRNA. Arrow indicates the time of the BMSC injection. *P < 0.05, compared with nontargeting siRNA control. n = 5 mice/group. (F and G) Dose-dependent reversal of mechanical allodynia by i.t. TGF-β1 at 5 and 21 days after CCI. Arrows in F and G indicate the time of the BMSC injection. *P < 0.05, compared with the vehicle group; #P < 0.05; n = 5 mice/group. (H) TGF-βR1 inhibitor SB431542 (100 pmol, i.t.) completely blocked the antiallodynic effect of TGF-β1 (10 ng, i.t.). Arrow indicates the time of i.t. injection given 19 days after CCI. *P < 0.05; n = 4–5 mice/group. Statistical significance was determined by 1-way ANOVA (A, B, and D), 2-way ANOVA, followed by Bonferroni’s post-hoc test (C, E, and FH), or Student’s t test (E and H). All data are expressed as the mean ± SEM. NT, nontargeting.
Figure 4
Figure 4. BMSCs administered i.t. inhibit CCI-induced glial activation and neuroinflammation in lumbar DRGs and the spinal cord dorsal horn.
(A) Paradigm showing the timing of BMSC treatment (CCI day 4) and tissue collection (CCI day 8). (B) Inhibition of CCI-induced upregulation of the macrophage marker IBA-1 in L4–L5 DRGs by i.t. injection of BMSCs (2.5 × 105 cells). Scale bar: 50 μm. (C) Quantification of IBA-1 staining. *P < 0.05, compared with the sham group; #P < 0.05; n = 4 mice/group. (D) qPCR showing expression levels of Gfap, Il1b, Il6, and Tnf mRNAs in L4–L5 DRGs and the effects of BMSCs. *P < 0.05, compared with the contralateral group; #P < 0.05; n = 4 mice/group. (E and F) BMSC inhibition of CCI-induced upregulation of the microglial marker IBA-1 and the astrocyte marker GFAP in the L4–L5 dorsal horn. Graph in F shows the quantification of GFAP and IBA-1 staining. Scale bar: 200 μm (top panels) and 50 μm (bottom panels). Bottom panels are enlarged images of the top panels. *P < 0.05, compared with the contralateral group; #P < 0.05; n = 4 mice/group. (G) qPCR showing the expression levels of Iba1, Il1b, Il6, and Tnf mRNAs and the effects of BMSCs. *P < 0.05, compared with the contralateral group; #P < 0.05; n = 4–5 mice/group. Statistical significance was determined by 1-way ANOVA, followed by Bonferroni’s post-hoc test. All data are expressed as the mean ± SEM.
Figure 3
Figure 3. BMSCs administered i.t. inhibit CCI-induced upregulation of ATF3 in DRGs and reduce CCI-induced downregulation of CGRP and IB4 in DRGs and the spinal cord dorsal horn.
(A) Paradigm showing the timing of BMSC treatment (CCI day 4) and tissue collection (CCI day 8). (BG) Inhibition of CCI-induced upregulation of ATF3 (B and C) and downregulation of IB4 and CGRP (D and E) in L4–L5 DRGs, as well as downregulation of IB4 and CGRP in the L4–L5 spinal cord dorsal horn (F and G) by i.t. injection of BMSCs (2.5 × 105 cells, 4 days after CCI). Scale bars: 50 μm (B, D, and F). Quantification results of ATF3 staining in DRGs (C), IB4 and CGRP staining in DRGs (E), and IB4 and CGRP staining in dorsal horns (G). *P < 0.05, compared with the sham or contralateral group; #P < 0.05; n = 4–5 mice/group. Statistical significance was determined by 1-way ANOVA, followed by Bonferroni’s post-hoc test. All data are expressed as the mean ± SEM.
Figure 2
Figure 2. Baseline pain and motor function are not altered by i.t. injection of BMSCs.
(AC) Baseline pain sensitivity evaluated by the von Frey test for mechanical sensitivity (A), the Hargreaves test for heat sensitivity (B), and the Randall-Selitto test for mechanical sensitivity. (D) Rotarod test for the evaluation of motor function. n = 6 mice/group. Arrows in AD indicate the time of BMSC injection.
Figure 1
Figure 1. Inhibition of CCI-induced evoked and ongoing neuropathic pain in mice by a single i.t. injection of BMSCs.
(A and B) Prolonged inhibition of mechanical allodynia (A) and thermal hyperalgesia (B) for 5 weeks by early treatment with i.t. injection of BMSCs (1.0 or 2.5 × 105 cells), given 4 days after CCI. *P < 0.05, compared with vehicle (PBS); n = 6 mice/group. (C and D) Reversal of mechanical allodynia (C) and thermal hyperalgesia (D) by late treatment with i.t. BMSCs (1.0 or 2.5 × 105 cells), given 14 days after CCI. Arrows in AD indicate the time of BMSC injection. *P < 0.05, compared with vehicle; #P < 0.05; n = 6 mice/group. (E) Paradigm for measuring ongoing pain using a 2-chamber CPP test. (F) CCI-induced ongoing pain was abolished by treatment with BMSCs. *P < 0.05, compared with saline; n = 5 mice/group. Statistical significance was determined by 2-way repeated-measures ANOVA, followed by Bonferroni’s post-hoc test (AD) or Student’s t test (F). All data are expressed as the mean ± SEM. BL, baseline.

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