Microvesicles shed from microglia activated by the P2X7-p38 pathway are involved in neuropathic pain induced by spinal nerve ligation in rats

Abstract

Microglia are critical in the pathogenesis of neuropathic pain. In this study, we investigated the role of microvesicles (MVs) in neuropathic pain induced by spinal nerve ligation (SNL) in rats. First, we found that MVs shed from microglia were increased in the cerebrospinal fluid and dorsal horn of the spinal cord after SNL. Next, MVs significantly reduced paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). In addition, the P2X7-p38 pathway was related to the bleb of MVs after SNL. Interleukin (IL)-1β was found to be significantly upregulated in the package of MVs, and PWT and PWL increased following inhibition with shRNA-IL-1β. Finally, the amplitude and frequency of spontaneous excitatory postsynaptic currents increased following stimulation with MVs. Our results indicate that the P2X7-p38 pathway is closely correlated with the shedding of MVs from microglia in neuropathic pain, and MVs had a significant effect on neuropathic pain by participating in the interaction between microglia and neurons.

Keywords: Microglia; Microvesicles; Neuropathic pain; Spinal nerve ligation, P2X7.

Fig. 1

The existence of microvesicles (MVs) in the cerebrospinal fluid (CSF) and dorsal horn of spinal cord of rats. a–c MVs were isolated from the CSF on different days following spinal nerve ligation (SNL) and analyzed by negative staining electron microscopy. Electron microscopy showed large vesicles with a morphology that was consistent with the characteristics of MVs, including delimitation by a lipid bilayer and a diameter larger than 100 nm. Scale bar = 100 nm. d–i Immunofluorescence labeling of MVs shed from microglia in the dorsal horn of spinal cord of sham group (d, f, h) and SNL 7-day rat (e, g, i). MVs derived from microglia were analyzed by fluorescence microscopy using the microglia marker IBA-1 (d, e) and the nucleus marker Hoechst (f, g). Merged images are shown in the right column (h, i). As indicated by arrows, MVs around microglia were stained by IBA1 without Hoechst (n = 4 rats for each group)

Fig. 2

a–e Flow cytometry analysis of microvesicles (MVs) presented in the rat cerebrospinal fluid after spinal nerve ligation, directly stained with IBA1 conjugated with FITC and Annexin V conjugated with APC. f The quantification of MVs stained with IBA-1 conjugated with FITC in flow cytometry experiments. All data are mean ± SEM. *P < 0.05, **P < 0.01, compared with control group. One-way ANOVA, followed by Bonferroni’s post hoc test was applied (n = 3 rats for each group)

Fig. 3

Time course of changes in paw withdrawal threshold and paw withdrawal latency following spinal nerve ligation (SNL) (a, b) or with injection of 10⁴ microvesicles (MVs) into naive rats (c, d) or 10 μL FTY720 (e, f 1 mM) into rats following SNL for 7 days. All data points are mean ± SEM. *P < 0.05, compared with control group or PBS group; n = 6 rats for each group. Statistical significance was determined by repeated measures ANOVA, followed by Student’s t test

Fig. 4

The number of microvesicles (MVs) in cerebrospinal fluid (CSF) decreased after FTY720 intrathecal injection on day 7 after SNL. a–c Flow cytometry analysis of MVs in CSF after PBS (a) or FTY720 injection 3 h (b) or FTY720 injection 6 h (c). d Quantity of IBA1⁺ MVs in a–c. All data are mean ± SEM. *P < 0.05, compared with PBS group, ^# P < 0.05; n = 3 rats for each group. Statistical significance was determined by one-way ANOVA, followed by Bonferroni’s post hoc test

Fig. 5

P2X7 receptors on microglia increased after spinal nerve ligation (SNL). a Western blot analysis of P2X7 receptors and p38 in spinal cord after SNL. b The quantitative analysis of P2X7 receptors and p38 normalized to β-actin at different times. c Fluorescent images of microglia from spinal cord in rats following SNL, stained for P2X7 receptors (red) and IBA-1 (green). Note the presence of many microvesicles at the cell surface positive for both P2X7 receptors and IBA1. All data points are mean ± SEM. *P < 0.05, a significant difference as compared with the sham group. One-way ANOVA, followed by Bonferroni’s post hoc test was applied. n = 4 rats for each group

Fig. 6

Inhibition of P2X7 receptors decreased the number of microvesicles (MVs) derived from microglia and alleviated pain after SNL. a P2X7 receptor expression level in ipsilateral spinal cord on after intrathecal injection of lenti-P2X7 siRNA or lenti-control siRNA. b, c Paw withdrawal threshold to mechanical stimuli (b) and paw withdrawal latency to thermal stimuli (c) of rats tested on day 7 after SNL, following intrathecal injection of lenti-P2X7 siRNA or lenti-control siRNA. d, e Flow cytometry analysis of MVs in cerebrospinal fluid (CSF) acquired after intrathecal injection of lenti-control siRNA (d) or lenti-P2X7 siRNA (e). f The quantification of IBA⁺ MVs in 100 μL CSF. Lenti-control siRNA or lenti-P2X7 siRNA was injected intrathecally 3 days before SNL, and spinal cord or CSF was acquired on day 7 after SNL. All data points are mean ± SEM. *P < 0.05, compared with basis level (BL) or control siRNA; ^# P < 0.05. (n = 6 rats for b and c, n = 3 rats for a, d, e, f)

Fig. 7

Hyperalgesia induced by microvesicles (MVs) was mediated, at least partly, by IL-1β. a, b Exposure to BzATP induced MV shedding from the N9 cell line (a, control; b, BzATP). c Concentration of IL-1β, TNF-α, and IL-6 in MVs obtained from the microglia cell medium by ELISA. d Concentration of IL-1β in MVs derived from N9 cells transfected with lenti-scrambled shRNA or lenti-IL-1β shRNA followed by stimulation with BzATP. e, f Paw withdrawal threshold to mechanical stimuli (e) and paw withdrawal latency to thermal stimuli (f) of rats administered with MVs (10⁴ per rat) obtained from N9 cells transfected with lenti-scrambled shRNA or lenti-IL-1β shRNA. Shedding MVs are indicated by an arrow in b. All data points are mean ± SEM. *P < 0.05, a significant difference as compared with the control group or the lenti-scrambled shRNA group. One-way ANOVA, followed by Bonferroni’s post hoc test (c) or Student’s t test (d) or repeated measures ANOVA, followed by Student’s t test (e, f) was applied. n = 3 independent cultures for c and d, and n = 6 rats for e and f

Fig. 8

Microglia-derived microvesicles (MVs) enhance excitatory neurotransmission. Representative recording of spontaneous excitatory postsynaptic current (sEPSC) amplitude and frequency in the dorsal horn of the spinal cord of sham rats (a) or spinal nerve ligation (SNL) 7 day rats (d) before and during MV treatment. Summary of the effect of MVs on the amplitude of sEPSC in sham rats (b) or SNL 7-day rats (e). Summary of the effect of MVs on the frequency of sEPSC in sham rats (c) or SNL 7-day rats (f). Two-tailed paired Student’s t test was applied to analyze the differences, with significance assumed at P < 0.05. Error bars are SEM (n = 3 rats for each group)