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Fig. 3 | The Journal of Headache and Pain

Fig. 3

From: P2X7R/NLRP3 signaling pathway-mediated pyroptosis and neuroinflammation contributed to cognitive impairment in a mouse model of migraine

Fig. 3

Repeated dural IS stimulation activated NLRP3 inflammasome and pyroptosis in the cortical and hippocampal neurons and microglia. (a-b) Representative immunoblots and quantification illustrated increased expression levels of NLRP3 (inflammasome receptor) and cleaved caspase-1 (marker of inflammasome activation) in the cerebral cortex after repeated dural IS stimulation. (c-e) Representative immunoblots and quantification illustrated increased expression levels of GSDMD-NT (pyroptotic marker) in the cerebral cortex after repeated dural IS stimulation. The difference in cleaved caspase-3 (apoptotic marker) did not reach a statistically significant level. (f-g) Double immunofluorescence staining showed that cleaved caspase-1 (CC1) and GSDMD were co-localized within the cortical and hippocampal neurons (NeuN positive) and microglia (Iba-1 positive), while cleaved caspase-3 (CC3) was co-localized within neurons (NeuN positive). Zoom magnification × 100. 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. Student’s t test was performed for statistical analysis. Data are represented as mean ± SEM. Abbreviations: IS, inflammatory soup; Cl. caspase-1, cleaved caspase-1; Cl. caspase-3, cleaved caspase-3; GSDMD-FL, full length Gasdermin D; GSDMD-NT, N-terminal Gasdermin D; CC1, cleaved caspase-1; CC3, cleaved caspase-3; NeuN, neuronal nuclei; Iba-1, ionized calcium-binding adaptor molecule-1; GFAP, glial fibrillary acidic protein 

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