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, 8 (1), 7848

N-methyl D-aspartate Receptor Subtype 2B Antagonist, Ro 25-6981, Attenuates Neuropathic Pain by Inhibiting Postsynaptic Density 95 Expression

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N-methyl D-aspartate Receptor Subtype 2B Antagonist, Ro 25-6981, Attenuates Neuropathic Pain by Inhibiting Postsynaptic Density 95 Expression

Ling-Er Huang et al. Sci Rep.

Abstract

Postsynaptic density-95 (PSD-95) is a synaptic scaffolding protein that plays a crucial role in the development of neuropathic pain. However, the underlying mechanism remains unclear. To address the role of PSD-95 in N-methyl-D-aspartate receptor subtype 2B (NR2B) -mediated chronic pain, we investigated the relationship between PSD-95 activation and NR2B function in the spinal cord, by using a rat model of sciatic nerve chronic constriction injury (CCI). We demonstrate that the expression levels of total PSD-95 and cAMP response element binding protein (CREB), as well as phosphorylated NR2B, PSD-95, and CREB, in the spinal dorsal horn, and the interaction of NR2B with PSD-95 were increased in the CCI animals. Intrathecal injection of the selective NR2B antagonist Ro 25-6981 increased paw withdrawal latency, in a thermal pain assessment test. Moreover, repeated treatment with Ro 25-6981 markedly attenuated the thermal hypersensitivity, and inhibited the CCI-induced upregulation of PSD-95 in the spinal dorsal horn. Furthermore, intrathecal injection of the PSD-95 inhibitor strikingly reversed the thermal and mechanical hyperalgesia. Our results suggest that blocking of NR2B signaling in the spinal cord could be used as a therapeutic candidate for treating neuropathic pain.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Spinal cord expression of NR2B and PSD-95 and behavioral signs of neuropathic pain induced by CCI (A). Western blot showing the expression of NR2B in the DH, VH, and DRG. Tubulin served as the loading control (B). Immunohistochemical localization of NR2B in the rat spinal cord. The line depicts dorsal- ventral axis. (CF1) Double immunostaining of NR2B (red; CF1) with PSD-95 (green; C), NeuN (green, a marker of neurons; D,D1), GFAP (green, a marker of astrocytes; E,E1), and Iba-1 (green, a marker of microglia; F,F1). The inset in C shows a magnification of the boxed area; (D1,E1,F1) are enlarged from (DF), respectively. Scale bars: (B), 100 μm; (CF1), 50 μm; inset in (C), 50 μm (G). Co-immunoprecipitation assays showing PSD-95 interactions with NR2B, P2X7R, and Homer1b/c in different nervous system regions (H,I). Quantification of thermal paw withdrawal latency (H) and paw withdraw mechanical threshold (I) 7 days after CCI- or sham-surgery. **P < 0.01 vs sham group (n = 7–8, Mann-Whitney test). Data are shown as mean ± SEM. CCI, chronic constriction injury; MC, motor cortex; ACC, anterior cingulate cortex; VH, ventral horn; DH, dorsal horn; DRG, dorsal root ganglion; IB, immunoblotting; IP, immunoprecipitation; Base, baseline.
Figure 2
Figure 2
Activation of NR2B/PSD-95/CREB signaling pathway following CCI. (AF) Representative uncropped western blots and the respective quantification of the immunoreactive bands, showing protein expression of NR2B (A), p-NR2B (B), PSD-95 (C), p-PSD-95 (D), CREB (E), and p-CREB (F) in the ipsilateral dorsal horn 7 days after CCI or sham operation. Blots were probed with specific antibodies, as indicated. Tubulin or GAPDH were used as loading controls, and run on the same blot. ***P < 0.001, **P < 0.001, *P < 0.05, n = 4–6, one-way ANOVA. (G,G1). Co-immunoprecipitation of PSD-95 with NR2B in the spinal dorsal horn after CCI (G) and quantification of the intensity of the immunoreactive bands (G1) Note that CCI significantly increases the PSD-95-NR2B interaction (*P = 0.022, n = 3–4, Mann-Whitney test). Data are shown as mean ± SEM and represent the fold-change compared to the values of the naïve sample. IB, immunoblotting; IP, immunoprecipitation; CCI, ipsilateral side to chronic constriction injury; Contra, contralateral side to chronic constriction injury.
Figure 3
Figure 3
Effect of Ro 25-6981 and NA-1 on paw withdrawal responses to noxious stimuli and on the expression of PSD-95 (AD). Quantification of thermal paw withdrawal latency (A,C) and paw withdrawal mechanical threshold (B,D) at different time-points after injection of DMSO or different concentrations of Ro 25-6981, as indicated. (***P < 0.001, **P < 0.01, *P < 0.05 vs. DMSO, n = 5–8, two-way ANOVA) (E). Representative uncropped western blots and quantification of the immunoreactive bands showing the effects of Ro 25-6981 on PSD-95 expression after CCI surgery. Tubulin served as the loading control and run on the same blot. The data are shown as the mean ± SEM and represent the fold-change compared to sham-operated mice. **P < 0.01 vs. sham, #P = 0.013 vs. CCI + DMSO, n = 5, one-way ANOVA (F,G). Quantification of thermal paw withdrawal latency (F) and paw withdrawal mechanical threshold (G) at different time-points after injection of NA-1 (125 ng/10 μL) 7 days after CCI surgery (*P < 0.05; **P < 0.01; ***P < 0.001 vs. NS ipsilateral, n = 5-6, two-way ANOVA). The data are shown as the mean ± SEM. CCI, chronic constriction injury; NS, vehicle; Ipsi, ipsilateral; Contra, contralateral; Ro, Ro 25-6981.

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References

    1. von Hehn CA, Baron R, Woolf CJ. Deconstructing the neuropathic pain phenotype to reveal neural mechanisms. Neuron. 2012;73:638–652. doi: 10.1016/j.neuron.2012.02.008. - DOI - PMC - PubMed
    1. Gilron I, Jensen TS, Dickenson AH. Combination pharmacotherapy for management of chronic pain: From bench to bedside. Lancet Neurol. 2013;12:1084. doi: 10.1016/S1474-4422(13)70193-5. - DOI - PubMed
    1. Ji RR, Xu ZZ, Gao YJ. Emerging targets in neuroinflammation-driven chronic pain. Nat Rev Drug Discov. 2014;13:533–548. doi: 10.1038/nrd4334. - DOI - PMC - PubMed
    1. Ji RR, Chamessian A, Zhang YQ. Pain regulation by non-neuronal cells and inflammation. Science. 2016;354:572–577. doi: 10.1126/science.aaf8924. - DOI - PMC - PubMed
    1. Yang Y, et al. Delayed activation of spinal microglia contributes to the maintenance of bone cancer pain in female Wistar rats via P2X7 receptor and IL-18. J Neurosci. 2015;35:7950–7963. doi: 10.1523/JNEUROSCI.5250-14.2015. - DOI - PMC - PubMed

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