Fig 2: Proposed role of inflammasome activation in increased neuronal excitability during CFA-evoked persistent pain in spinal dorsal horn. CFA treatment induces peripheral inflammation in skin. Inflammatory mediators secreted by the local cells sensitize the nociceptive nerve fiber terminals and, in turn, the primary afferent fibers, terminating in the superficial laminae of the spinal dorsal horn release substances which activate astrocytes. When mechanical pain sensitivity is highest, the NLRP2 inflammasome sensor and IL-1β is overexpressed in astrocytes. Our previous findings suggest that the major target of the secreted, cleaved IL-1β is spinal neurons. IL-1R1 ligand binding affects neuronal excitability via interacting NMDA and AMPA receptor-mediated glutamate signaling, which can lead to activation of the local neuronal networks and the nociceptive sensory pathways.

Fig 3: Distance measurement of NLRP markers from astrocyte profiles. Illustrations showing IMARIS rendered confocal double-stained z-stack images with putative distance (0–5µm) of NLRP1 (a), NLRP2 (b) and NLRP3 (c) inflammasomal proteins (colorful spots, indicated by a color scale) from GFAP-positive astrocyte profiles (red). In the case of NLRP1 and NLRP3, no significant changes were detected in distance distribution between control (CTRL) and chronic inflammatory pain (CFA); however, for the NLRP2 marker, a significant increase (p = 0.0004) in spot number was calculated in the range of 0–1 µm in chronic pain in comparison with control. Scale bars: 10 μm.

Fig 4: NLRP3 activation in microglia was associated with surgery-induced inflammation. NLRP3 activation in microglia was associated with surgery-induced inflammation. (A): The expression levels of NLRP1, AIM2, NLRP2, NLRP3, and NLRC4 in the hippocampus were examined by Immunoblotting, and the statistical data are shown in (B) (means ± SEM, n = 3). (C): pro-IL1β/IL1β,pro-CASP1/CASP1/CASPASE1 protein expression in the hippocampus were measured by Immunoblotting and the quantitative analysis data shown in (D) (means ± SEM, n = 3). (E,F) Immunofluorescence staining of IBA-1, GFAP and NLRP3 in the hippocampal dentategyrus brain region. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the corresponding sham group, as determined by the Student’s t-test.

Fig 5: Astrocytic NLRP2 expression, but not NLRP3, is significantly increased during CFA-evoked inflammatory pain. (a) Histogram shows that the CFA-evoked inflammation induced an increase in the degree of co-localization between GFAP and NLRP2 markers in the superficial spinal dorsal horn of rats. Columns indicate the percentage of GFAP profiles that were found to also be labeled with NLRP2. White column shows data obtained from control animals (day 0), whereas the black column represents values found in CFA-injected animals 3 days after CFA injection into the right hind paw (day 3). CFA-evoked inflammation significantly increased the proportion of NLRP2 immunoreactive spots on astrocyte profiles (p = 0.000000148). Data are shown as mean ± SEM. (b) Histogram with the same key shows that CFA injection did not cause significant changes in the degree of co-localization between GFAP and NLRP3 markers in the superficial spinal dorsal horn of rats. Data are shown as mean ± SEM. (c) Histogram with the same key showing a significant enhancement of NLRP2 immunoreactive spots in chronic inflammatory pain compared to control (100%) (p = 0.000000111). Data are shown as mean ± SEM. (d) Western blot analysis shows substantial increase of NLRP2 protein in tissue lysates of spinal cord 3 days after CFA administration (CFA-ipsi day 3) compared to control (CTRL). NLRP1 and NLRP3 proteins are also detectable in the tissue samples. *** p < 0.001.