Fig. 2

Supraphysiological doses of 17β-Estradiol (sE2) exacerbate microglial response and neuroinflammation in the brains of ovariectomized mice. Immunohistochemical staining is used to label microglia in paraffin sections of mouse brain tissues with an anti-Iba1 antibody. Representative pictures (A) and quantification of the cell body area and endpoints of Iba1-labeled microglia in each group of mice (B, C). Bar = 5 µm. D Areas for immunofluorescence analysis marked on HE-stained coronal brain sections. The hippocampus is marked in yellow squares and the cortex is marked in red squares. E Typical images of microglia (Iba1-labeled) and M1 polarization markers (i.e., CD86, IL-1β, IL-6, and TNF-α) co-located by immunofluorescence. Bar = 50 µm. F Quantification of the number of positive cells for microglia M1 polarization markers per field in each group of mice. The mRNA levels of proinflammatory factors IL-1β, IL-6, and TNF-α (G), and anti-inflammatory factors IL-4, IL-10, and TGF-β (H) are evaluated by qPCR analysis. Student’s t-test is performed to determine the significant difference based on P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***), respectively, in comparison to the sham group (as indicated by black asterisks and “ns”) or the OVX group (as indicated by red asterisks). ns: no statistical significance. Data are presented as mean ± the standard deviation (SD) of at least four animals per group