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. Author manuscript; available in PMC: 2022 Sep 1.
Published in final edited form as: Toxicology. 2021 Aug 17;461:152901. doi: 10.1016/j.tox.2021.152901

Fig. 3.

Fig. 3

(A.) Colocalization events of CD31 (red) and Claudin5 (green) immunoreactivity in brain sections from Chow, Chow+MC, MCD-HFD, MCD-HFD+MC, and MCD-HFD+MC/NLRP3KO mice groups as shown by immunofluorescence microscopy. Colocalization of CD31 and Claudin5 was represented by yellow dots and highlighted by white circles. All images were taken at × 40 magnification (Scale: 50 μm). (B.) Colocalization events of CD31 (green) and ZO1 (red) and Immunoreactivity of MMP9 expression in Chow, Chow+MC, MCD-HFD, MCD-HFD+MC, and MCD-HFD+MC/NLRP3KO mice groups as depicted by immunofluorescence microscopy and immunohistochemistry, respectively. Colocalization of CD31 and ZO1 was represented by yellow dots and highlighted by white circles whereas MMP9 immunoreactivity was indicated by black arrows. Morphometric analysis of (C.) Claudin5/CD31 and ZO1/CD31 colocalization events, (D.) MMP9 immunoreactivity [mean data plotted on y-axis was measured as % positive immunoreactive area (% ROI) in arbitrary light units from three different microscopic fields] in Chow, Chow+MC, MCD-HFD, MCD-HFD+MC, and MCD-HFD+MC/NLRP3KO mice groups (*p< 0.05, ***p< 0.001). (E.) Western blot analysis of ZO1, Claudin5 protein expression levels in the mouse primary brain endothelial cell lysates. Lanes 1–5 represent untreated cells, MC+Lcn2, MC+Lcn2+CRID3, MC, and MC+CRID3 groups of cells, respectively. (F.) Band quantification of ZO1, and Claudin5 immunoblot normalized against β-actin (*p< 0.05, **p< 0.01, ns=non-significant). (G.) IL −1β level (pg/mL) was detected by ELISA in supernatants obtained from untreated cells, MC, MC+CRID3, Lcn2+MC, and MC+Lcn2+CRID3 groups of mouse primary brain endothelial cells and displayed by bar graph. (***p< 0.001) (H.) S100B level (pg/mL) was quantified by ELISA using serum from Chow, Chow+MC, MCD-HFD, MCD-HFD+MC, and MCD-HFD+MC/NLRP3KO mice groups and plotted as a bar graph (**p< 0.01). All statistical significance was tested by performing unpaired t-test between the groups (*p< 0.05, **p< 0.01, ***p< 0.001, ns=non-significant), followed by Bonferroni Dunn Post hoc corrections.