Fig. 2

Neuropathic pain injury induces glial activation and neuroinflammation in the ipsilateral spinal cord. A Pain sensitivity was assessed using the von Frey filament test to measure mechanical thresholds from baseline (day 0) to day 14 post-injury (two-way ANOVA, ****p < 0. 001, n = 5 mice per group) B-E Representative confocal images of the ipsilateral superficial dorsal horn (laminae I/II) in the sham and SNL groups. Tissues were stained for IBA1 (microglia, B), GFAP (astrocytes, B), and c-Fos (neurons, D), with data presented at both low (left, scale bar = 50 μm) and high magnification (right, scale bar = 20 μm). Quantification of the mean intensity of IBA1 and GFAP (C) and the number of c-Fos-positive cells (E) in the sham and SNL groups (unpaired t-test, ***p < 0.001, ****p < 0.0001, error bars: SEM, n = 5 mice per group). F The ipsilateral dorsal horn of sham and SNL rats was analyzed for mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, iNOS, and IL-4) via qRT-PCR (unpaired t-test, *p < 0.05, **p < 0.01, ***p < 0.001, error bars: SEM, n = 5–6 mice per group). G, I, K Representative confocal images of the contralateral (Contra) and ipsilateral (Ipsi) dorsal and ventral horns of the spinal cord in SNL rats. Tissues were stained for IBA1 (G), GFAP (I), and c-Fos (K), with dashed lines indicating the laminae (I-VI) layers of the spinal cord. H, J Quantification of the mean intensity of IBA1 (H) and GFAP (J) (unpaired t-test, *p < 0.05, **p < 0.01, ***p < 0.001, error bars: SEM, scale bar = 250 μm, n = 5–6 mice per group). L Comparison of the number of c-Fos-positive cells between superficial (I/II) and deep layers (III/IV/V/VI) in each ipsilateral and contralateral side (unpaired t-test, n.s. = nonsignificant, error bars: SEM). Data are representative of n = 5–6 independent experiments