Spinal inhibition of p38 MAP kinase reduces inflammatory and neuropathic pain in male but not female mice: Sex-dependent microglial signaling in the spinal cord

Abstract

Previous studies have shown that activation of p38 mitogen-activating kinase (MAPK) in spinal microglia participates in the generation of inflammatory and neuropathic pain in various rodent models. However, these studies focused on male mice to avoid confounding effects of the estrous cycle of females. Recent studies have shown that some spinal pro-inflammatory signaling such as Toll-like receptor 4-mediated signaling contributes to pain hypersensitivity only in male mice. In this study we investigated the distinct role of spinal p38 in inflammatory and neuropathic pain using a highly selective p38 inhibitor skepinone. Intrathecal injection of skepinone prevented formalin induced inflammatory pain in male but not female mice. Furthermore, intrathecal skepinone reduced chronic constriction injury (CCI) induced neuropathic pain (mechanical allodynia) in male mice on CCI-day 7 but not CCI-day 21. This male-dependent inhibition of neuropathic pain also occurred in rats following intrathecal skepinone. Nerve injury induced spinal p38 activation (phosphorylation) in CX3CR1-GFP(+) microglia on CCI-day 7, and this activation was more prominent in male mice. In contrast, CCI induced comparable microgliosis and expression of the microglial markers CX3CR1 and IBA-1 in both sexes. Notably, intraperitoneal or local perineural administration of skepinone inhibited CCI-induced mechanical allodynia in both sexes of mice. Finally, skepinone only reduced the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in lamina IIo neurons of spinal cord slices of males 7days post CCI. Therefore, the sex-specific p38 activation and signaling is confined to the spinal cord in inflammatory and neuropathic pain conditions.

Keywords: Inflammatory pain; MAPK; Male; Microglia; Neuropathic pain; Sex; p38.

Figure 1. Spinal or systemic inhibition of p38 signaling inhibits spontaneous pain behavior in male but not female mice following formalin injection

(A–C) IT injection of the p-p38 inhibitor skepinone, 30 minutes prior to intraplantar administration of formalin, dose-dependently reduced spontaneous Phase II pain behavior in male but not female mice. (A,B) Time course of formalin-induced spontaneous pain in males (A) and females (B). (C) Formalin-induced Phase I (0–10 min) and Phase II (15–45 min) responses. *P< 0.05, **P< 0.01, in comparison to same sex saline control, One-Way ANOVA, n = 6–8 mice per sex per group. n.s., not significant. (D,E) IP administration of skepinone 1 hour prior to formalin injection reduced spontaneous Phase II pain behavior in male but not female mice. (D) Time course of formalin-induced spontaneous pain in males and females. (E) Formalin-induced Phase I (0–10 min) and Phase II (15–45 min) responses. *P< 0.05, **P< 0.01, in comparison to same sex saline control. ^#P< 0.05, compared to opposite sex. One-Way ANOVA, n = 6–8 mice per sex per group.

Figure 2. Spinal inhibition of p38 inhibits CCI-induced mechanical allodynia in male mice and rats but not in female mice and rats one week after nerve injury

(A) IT injection of 30 μg per mouse of the p-p38 inhibitor skepinone 7 days post-CCI significantly increased paw withdrawal threshold in males but not female mice. **P<0.01, in comparison to same sex vehicle (saline). ^#P<0.05 in comparison to opposite sex drug, Two-Way ANOVA, repeated measures, n = 6 mice per sex per group. (B) IT injection of 30 μg per mouse of the p-p38 inhibitor skepinone 21 days post-CCI had no effects on mechanical allodynia in both male and female mice. P>0.05, Two-Way ANOVA, repeated measures, n = 5 mice per sex per group. (C) IT injection of 60 μg per rat of the p-p38 inhibitor skepinone 7 days post-CCI significantly increased paw withdrawal threshold in male but not female rats. *P< 0.05, in comparison to same sex. ^#P<0.05 in comparison to opposite sex, Two-Way ANOVA, repeated measures, n = 6 rats per sex per group.

Figure 3. Systemic or peri-neural (peri-sciatic) inhibition of p38 signaling decreases allodynia in both sexes

(A) IP administration of 30 mg/kg skepinone 7 days post-CCI reduced mechanical allodynia in both male and female mice. *P<0.05, **P<0.01, ***P<0.001, in comparison to same sex pre-injection control. Two-Way ANOVA, repeated measures, n = 6 mice per sex per group. n.s., not significant. (B) Peri-sciatic (peri-neural) administration of 18 μg per mouse skepinone 7 days post-CCI reduced mechanical allodynia in both sexes. *P<0.05, **P<0.01, ***P<0.001, in comparison to same sex saline control. Two-Way ANOVA, repeated measures, n = 6 mice per sex per group. n.s., not significant.

Figure 4. Spinal dorsal horn microglia display morphological activation, increased IBA-1 staining intensity, and proliferation in response to CCI in both sexes

(A,B) Immunofluorescent staining with the microglial marker IBA-1 (red) in lumbar dorsal horn sections showing both contralateral (Contra) and ipsilateral (Ipsi) sides of male (A) and female (B) mice 7 days post-CCI. Scales, 50 and 100 μm. (C) Quantification IBA-1 immunofluorescence from mouse lumbar sections shows a significant increase in fluorescent intensity and the number of IBA-1 positive cells in the ipsilateral dorsal horn in both male and female mice. *P<0.05, ***P< 0.001, comparing ipsilateral and contralateral dorsal horn of same sex. Two-tailed Student t-test, n = 3 mice per sex per group, 3 sections per mouse.

Figure 5. Nerve injury induces p38 phosphorylation in spinal cord dorsal horn of male but not female mice

(A,B) Western blots of p-p38 and p38 from male (A) and female (B) contralateral (Contra) and ipsilateral (Ipsi) lumbar dorsal horn samples (L4–6) obtained 7 days post-CCI. GAPDH was used as a loading control. (C,D) Quantification of the intensity of p-p38 (C) and p38 (D) western bands shows that p-p38 and p38 levels are significantly increased in male but not female ipsilateral dorsal horn compared to contralateral (*P< 0.05) or female ipsilateral dorsal horn (^#P <0.05). Values were normalized to GAPDH as a loading control then to the contralateral dorsal horn. Two-tailed Student t-test, n = 4 mice per sex per group. (E) Quantification of the ratio of p-p38/p38 western bands in ipsilateral and contralateral dorsal horn of males and females. *P< 0.05, compared to contralateral side, n=4 mice per sex per group. n.s., not significant.

Figure 6. Nerve injury induces spinal CX3CR1 expression in both sexes and spinal microglial p38 activation primarily in male mice 7 days post CCI

(A, B) CX3CR1-GFP (green) and immunofluorescent staining of p-p38 (red) in dorsal horn sections from CX3CR1-GFP mice of a male (A) and a female (B) 7 days post-CCI. GFP shows morphological activation and proliferation of microglia in the dorsal horn of both male and female mice. Note that p-p38 staining is colocalized with the microglial marker CX3CR1. Also note that p-p38 staining is stronger in male microglia. Scales, 50 μm (up panel in B) and 25 μm (low panel in B).

Figure 7. Nerve injury induces spinal microglial proliferation in both sexes and spinal microglial p38 activation primarily in male mice 7 days post CCI

(A) Quantification from lumbar spinal cord sections of CX3CR1-GFP mice shows a significant increase in the number of GFP positive microglia in the medial superficial dorsal horn ipsilateral to CCI in both male and female mice. ***P< 0.001 comparing ipsilateral and contralateral of same sex, two-tailed Student t-test, n = 3 mice per sex per group. n.s., not significant. (B) Quantification of p-p38 positive cells in the medial superficial dorsal horn from lumbar spinal cord sections shows a significant increase in the number of p-p38 positive cells in in both male and female mice. *P<0.05, comparison between ipsilateral and contralateral of same sex, n = 4 mice per sex per group. n.s., not significant. (C) Quantification of p-p38 immunofluorescence in the medial superficial dorsal horn from lumbar spinal cord sections shows a significant increase in intensity of p-p38 immunofluorescence in both male and female mice and further increase in male mice. *P<0.05, comparison between ipsilateral and contralateral of same sex; ^#P<0.05, male vs. female, two-tailed Student t-test, n = 4 mice per sex per group. Four-five spinal cord sections were included per mouse for quantification.

Figure 8

Skepinone inhibits the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in lamina IIo neurons of spinal cord slices in male but not female mice 7 days post CCI. (A) Traces of sEPSCs in lamina IIo neurons with and without skepinone (3 μM) treatment. (B) Frequency and amplitude of sEPSCs, *P<0.01, two-tailed Student t-test, n=5 neurons/group.