Fig 2: Repeated dural IS stimulation upregulated P2X7R in the cortical and hippocampal neurons. (a-b) Representative immunoblots and quantification illustrated increased levels of P2X7R in the cerebral cortex and hippocampus after repeated dural IS stimulation. (c-e) Double immunofluorescence staining showed that P2X7R was co-localized within neurons (NeuN positive) in the cerebral cortex and hippocampus. No substantial colocalization of P2X7R was observed in microglia (Iba-1 positive) and astrocytes (GFAP positive) in the cortex and hippocampus. Magnification × 40. Insets show a higher magnification view (Zoom). Scale bar, 20 μm. Images were taken under identical exposures and conditions. n = 6 mice per group. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared to sham mice. Datasets in (b) were analyzed using Student’s t test. Data are represented as mean ± SEM. Abbreviations: IS, inflammatory soup; NeuN, neuronal nuclei; Iba-1, ionized calcium-binding adaptor molecule-1; GFAP, glial fibrillary acidic protein

Fig 3: Inhibition of P2X7R attenuated IS-induced gliosis and neuronal loss in the cerebral cortex and hippocampus. (a, b, e, f) Representative immunofluorescence and quantification of Iba-1 and GFAP illustrating resistance to microgliosis and astrogliosis due to decreased expression of Iba-1and GFAP in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. Magnification × 40. Insets show a higher magnification view. Scale bar, 20 μm. (c, g) Representative immunofluorescence and quantification of MAP2 illustrating resistance to neuronal loss due to decreased expression of MAP2 in the cerebral cortex and hippocampus of IS-BBG mice compared to IS-VEH mice. Magnification × 20. Scale bar, 20 μm. (d, h) Representative immunofluorescence and quantification of MBP illustrating no significant difference in white-matter integrity in the cerebral cortex and hippocampus among the four groups. Magnification × 20. Scale bar, 20 μm. All images were taken under identical exposures and conditions. n = 6 mice per group. *p < 0.05, **p < 0.01 and ***p < 0.001 as compared to sham-VEH mice; +p < 0.05, ++p < 0.01 and +++p < 0.001 as compared to IS-VEH mice. One-way ANOVA with Tukey’s multiple comparisons or Kruskal–Wallis with Dunn’s multiple comparisons test were performed for statistical analysis. Data are represented as mean ± SEM. Abbreviations: IS, inflammatory soup; BBG, brilliant blue G; VEH, vehicle; Iba-1, ionized calcium-binding adaptor molecule-1; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; MBP, myelin basic protein