p38 phosphorylation in medullary microglia mediates ectopic orofacial inflammatory pain in rats

Abstract

Background: Orofacial inflammatory pain is likely to accompany referred pain in uninflamed orofacial structures. The ectopic pain precludes precise diagnosis and makes treatment problematic, because the underlying mechanism is not well understood. Using the established ectopic orofacial pain model induced by complete Freund's adjuvant (CFA) injection into trapezius muscle, we analyzed the possible role of p38 phosphorylation in activated microglia in ectopic orofacial pain.

Results: Mechanical allodynia in the lateral facial skin was induced following trapezius muscle inflammation, which accompanied microglial activation with p38 phosphorylation and hyperexcitability of wide dynamic range (WDR) neurons in the trigeminal spinal subnucleus caudalis (Vc). Intra-cisterna successive administration of a p38 mitogen-activated protein kinase selective inhibitor, SB203580, suppressed microglial activation and its phosphorylation of p38. Moreover, SB203580 administration completely suppressed mechanical allodynia in the lateral facial skin and enhanced WDR neuronal excitability in Vc. Microglial interleukin-1β over-expression in Vc was induced by trapezius muscle inflammation, which was significantly suppressed by SB203580 administration.

Conclusions: These findings indicate that microglia, activated via p38 phosphorylation, play a pivotal role in WDR neuronal hyperexcitability, which accounts for the mechanical hypersensitivity in the lateral facial skin associated with trapezius muscle inflammation.

Fig. 1

Changes in mechanical sensitivity following CFA injection into trapezius muscle. HWT measured in facial skin for 15 days after CFA or saline injection into the ipsilateral trapezius muscle. The inset indicates the stimulus site. *p < 0.05 compared with saline-injected rats. Data represent mean ± SEM. (n = 10 in each group; two-way ANOVA with repeated measures, followed by Bonferroni’s multiple-comparison tests).

Fig. 2

Microglial activation in Vc following CFA injection into trapezius muscle. a Photomicrograph of Iba1-IR cells in Vc on day 4 after the CFA injection. High-magnificated photomicrograph of Iba1-IR cells in Vc on day 4 after CFA (b) or saline (c) injection. d High-magnificated photogmicrograph of Iba1-IR cells in Vc on day 15 after CFA injection. Scale bar 500 μm (a); 100 μm (b–d). e Density of the Iba1 immuno-products in Vc (1,440, 2,160 and 2,800 μm caudal to the obex) on day 4 and 15 after saline or CFA injection. Data represent mean ± SEM; n = 13–14 in each; *p < 0.05, **p < 0.01; compared to saline-injected rats by Student’s t tests.

Fig. 3

p38 phosphorylation in microglia in Vc. Photomicrographs of pp38-IR cells (a, d, g), Iba1-IR cells (b), pp38-IR and Iba1-IR cells (c), GFAP-IR cells (e), pp38-IR and GFAP-IR cells (f), NeuN-IR cells (h), and pp38-IR and NeuN-IR cells (i) in Vc on day 4 after CFA injection. The arrow denotes double-IR cells. j Relative amount of pp38 protein in Vc on day 4 after CFA or saline injection. p38 protein was used as a loading control. Data represent mean ± SEM; n = 12 in each; ***p < 0.001; compared to saline-injected rats by Student’s t tests.

Fig. 4

Mechanical sensitivity in facial skin and microglial activation in CFA-injected rats with i.c.m. SB203580 administration. a Relative amount of pp38 protein in Vc on day 4 in saline or CFA-injected rats with i.c.m. vehicle or SB203580 administration. p38 protein was used as loading control. Data represent mean ± SEM. n = 16–17 in each. *p < 0.05, **p < 0.01; by one-way ANOVA followed by Tukey’s multiple-comparison tests. b HWT measured in facial skin on day 4 after CFA or saline injection with i.c.m. vehicle or SB203580 administration. n = 9–10 in each. **p < 0.01; by one-way ANOVA followed by a Kruskal–Wallis tests. Photomicrograph of Iba1-IR cells on day 4 after CFA injection into the trapezius muscle with i.c.m. vehicle (c) or SB203580 (d) administration. e Photomicrograph of Iba1-IR cells on day 4 after saline injection into the trapezius muscle with i.c.m. vehicle administration. Scale bar 100 μm. f Density of Iba1 immuno-products in Vc on day 4 in CFA- or saline-injected rats with i.c.m. vehicle or SB203580 administration. Data represent mean ± SEM. n = 9–10 in each. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; by one-way ANOVA followed by Tukey’s multiple-comparison tests.

Fig. 5

IL-1β protein in saline- or CFA-injected rats with i.c.m. vehicle or SB203580 administration. Photomicrographs of IL-1β-IR cells (a, d, g), Iba1-IR cells (b), IL-1β-IR and Iba1-IR cells (c), GFAP-IR cells (e), IL-1β-IR and GFAP-IR cells (f), NeuN-IR cells (h), and IL-1β-IR and NeuN-IR cells (i) in Vc on day 4 after CFA injection. The arrow indicates double-IR cells. j Relative amount of IL-1β protein in Vc on day 4 after CFA or saline injection with i.c.m. vehicle or SB203580 administration. β-Actin protein was used as loading control. Data represent mean ± SEM. n = 13 in each. *p < 0.05, **p < 0.01; by one-way ANOVA followed by Tukey’s multiple-comparison tests.

Fig. 6

Mechanical-evoked responses of Vc neurons after CFA injection with i.c.m. SB203580 administration. a Background activities of WDR neurons. WDR neuronal responses to mechanical stimuli by brush, pinch stimuli (b) or von Frey filament (c) on day 4 after saline or CFA injection with i.c.m. vehicle or SB203580 administration (9 WDR neurons from 4 CFA-injected rats with i.c.m. vehicle administration, 9 WDR neurons from 4 CFA-injected rats with i.c.m. SB203580 administration, 9 WDR neurons from 3 saline-injected rats with i.c.m. vehicle administration). Data represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; by one-way ANOVA followed by Tukey’s multiple-comparison tests or by two-way ANOVA with repeated measures followed by Tukey’s multiple-comparison tests.

Fig. 7

Schematic presentation of contribution of p38 phosphorylation in medullary microglia to ectopic orofacial inflammatory pain. Microglia in medulla and upper cervical spinal cord was activated following trapezius muscle inflammation. IL-1β release from the activated microglia in trigeminal spinal subnucleus caudalis (Vc) was accelerated through p38 phosphorylation and the excitability of wide dynamic range (WDR) neurons in Vc was enhanced via IL-1β signaling. The enhancement of excitability of WDR neurons in Vc after trapezius muscle inflammation may result in ectopic mechanical allodynia in orofacial region.