SHED-CM prevents the partial sciatic nerve ligation-induced pain
We tightly ligated 1/3 to 1/2 of the SCN on the right side in mice to induce NP. A decrease in the threshold for tactile stimuli was observed after nerve ligation. The threshold was reached in a minimum of 3 days after PSL and was maintained at the level for weeks. Daily intravenous administration of SHED-CM, immediately after PSL, inhibited PSL-induced mechanical allodynia (Figs. 1a and b; early phase). These antinociceptive effects of SHED-CM were detected even 3 days after the administration of SHED-CM. In the von Frey test, the threshold for the right hindpaw at day 3 was 6.73 ± 1.5 g in the SHED-CM group and 4.28 ± 1.48 g in the DMEM group. On day 7, the threshold of SHED-CM group was 8.39 ± 0.99 g, which was significantly higher than 4.29 ± 1.77 g in the DMEM group. In contrast, no antinociceptive effects were observed in the Fibro-CM group (Fig. 1b). We also examined whether SHED-CM could attenuate PSL-induced pain in a well-developed phase (middle phase). SHED-CM treatment exhibited significant antinociceptive effects in the middle phase model, in which the threshold of the SHED-CM group was 8.07 ± 0.84 g and of the DMEM group was 4.82 ± 0.92 g at 14 days after PSL (Figs. 1c and d). In the late phase, the threshold of the SHED-CM group was 9.47 ± 0.74 g and that of the DMEM group was 5.07 ± 0.67 g at 21 days after PSL (Figs. 1e and f). During the SHED-CM treatment, the von Frey test data of the contralateral side did not show any change (Additional file 3: Suppl. Fig 2). None of the test groups showed signs of motor weakness during the experimental period.
SHED-CM treatment induces M2-polarized macrophages in PSL
To investigate the analgesic mechanism of SHED-CM, we examined the mRNA expression profiles of genes involved in pro- and anti-inflammatory responses in the early phase PSL. Seven days after PSL, the expression of inflammatory genes, TNF-α, IL-1β, and inducible nitric oxide synthase (iNOS), was greatly increased, but was markedly suppressed by the SHED-CM treatment. In contrast, the SHED-CM treatment increased the expression of pan macrophage markers, F4/80 and M2-specific molecules, CD206, arginase-1 (Arg-1), and Ym-1. Notably, we found that the SHED-CM treatment elevated the expression of an array of neurotrophic and immunosuppressive factors, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell derived neurotrophic factor (GDNF), and transforming growth factor-β1 (TGF-β1) (Fig. 2). These results show that the SHED-CM treatment converted the proinflammatory microenvironment of PSL to anti-inflammatory and tissue-protective one.
Immunohistochemical analysis revealed that the number of CD206+ F4/80+ macrophages was significantly increased, but TNF-α+ S100+ proinflammatory SCs were reduced by the SHED-CM treatment (Figs. 3a-d).
SHED-CM treatment induces M2-polarized macrophages in ipsilateral DRG
Immunohistochemical analysis of the ipsilateral L4/L5 DRG, 7 days after PSL, revealed that the SHED-CM treatment significantly increased the number of CD206+ F4/80+ M2 macrophages compared with the DMEM control treatment (Fig. 3e). The cell count analysis showed that the number of M2 cells in the SHED-CM group was significantly higher than that in the DMEM group (Fig. 3f), indicating that M2 not only accumulated in the ipsilateral SCN, but also in the ipsilateral DRG.
SHED-CM attenuates PSL-induced microglial activation in the spinal cord
Next, we examined microglial activation, 7 days after PSL. As shown in Fig. 3g, a substantial increase in the number of Iba1+ microglia in the L3/4 ipsilateral dorsal horn was obvious compared with that in the contralateral horn. Microglial morphology in the ipsilateral side showed activated morphology with a hypertrophied soma and thicker and retracted processes, whereas that of the contralateral side seemed to be at the quiescent stage with smaller soma and ramified processes; the two kinds of morphologies are very distinguishable (43). Notably, in the SHED-CM-treated group, the number of Iba1+ microglia on the ipsilateral side was reduced, and their morphologies were very similar to those on the contralateral side (Fig. 3h). These results demonstrate that SHED-CM treatment suppressed the PSL-induced microglial activation in the spinal cord.
M2 macrophages induced by SHED-CM are required for its anti-nociceptive activity
Next, we investigated the roles of M2 macrophages in the antinociceptive activity of SHED-CM by specifically depleting them with m-Clo. After PSL, SHED-CM was injected daily for 7 consecutive days, and from day 4 to day 6, m-Clo or m-Enc was injected together with SHED-CM (Fig. 4a).
The antinociceptive effects were similar in both the groups at day 3; however, m-Clo, but not m-Enc, significantly decreased at day 5. On day 7, the von Frey test of the m-Clo, m-Enc, and DMEM groups was 5.49 ± 0.92 g, 7.9 ± 1.33 g, and 4.57 ± 1.00 g, respectively (Fig. 4b). None of the test groups showed signs of motor weakness during the experimental period.
Immunohistochemical analysis showed that m-Clo, but not m-Enc, reduced the number of CD206+ F4/80+ macrophages in SCN and DRG, while increasing TNF-α+ S100+ proinflammatory SCs in SCN and Iba1+ activated microglia in the spinal cord (Figs. 5a-h). These results show that SHED-CM-induced M2 macrophages are crucial for the suppression of proinflammatory response and antinociceptive activity.
CM from M2 induced by SHED-CM suppressed proinflammatory activities of Schwann cells in vitro
We next examined the biological activity of the secretion from M2 induced by SHED-CM. MCSF-treated bone marrow cells differentiated into macrophages, which were subsequently stimulated with SHED-CM for 24 h. Using this procedure, more than 68.48% of the cells differentiated into CD206+F4/80+M2 macrophages (Fig. 6a). We harvested the secretion from M2 induced by SHED-CM as M2-CM.
SCs first detect nerve injury and play a critical role in the development and maintenance of NP. Under proinflammatory conditions, they express cytokines TNF-α and IL-1β, chemokine MCP-1, and transient receptor potential ankyrin 1 (TRPA 1) channels, which accelerate neuroinflammation and mechanical allodynia. We treated human SCs with TNF-α or M2-CM for 24 h and analyzed the gene expression profile. The TNF-α treatment increased the expression of TRPA1, TNF-α, IL-1β, and MCP-1, whereas the M2-CM treatment strongly suppressed this upregulation (Fig. 6b).
SHED-CM derived M2-CM attenuates neuropathic pain in vivo
To examine the antinociceptive activity of M2-CM, we administered it to the PSL mice. We found that M2-CM, but not DMEM, prevented PSL-induced allodynia and proinflammatory responses in the SCN (Figs. 7a-c). On the ipsilateral side of L3/4, IBA1+ positive cells were extensively decreased in the M2-CM group compared with that in the DMEM group (Figs. 7d and e). Taken together, these results suggest that SHED-CM suppressed the neuroinflammation and mechanical allodynia in part through the analgesic effect of M2.
The therapeutic factors in SHED-CM attenuate neuropathic pain in vivo
To confirm the therapeutic effects of a set of M2 inducers, MCP-1 and sSiglec-9, in SHED-CM, we intravenously administered them to the PSL mice. We found that, in the middle phase setting, the von Frey test of the right hindpaw of the MCP-1/sSiglec-9 group at day 14 was 7.32 ± 1.75 g, which was significantly higher than that of the DMEM and PBS groups but was lower than that of the SHED-CM group (Figs. 7f and g). These results suggest that the promising analgesic ability of SHED-CM may partly rely on MCP-1/sSiglec-9.