Fig. 6

Supraphysiological doses of 17β-Estradiol (sE2) exacerbate neuronal damage in ovariectomized mice. Representative images of HE (left) and Nissl (right) staining of coronal brain sections. Bar = 50 µm. Black, light blue, and dark blue boxes indicate the hippocampal CA1, CA2, and DG areas; green boxes indicate the cortex. B Quantification of total neuron (up) and cell death by Nissl staining in neurons (down). C Viability of HT22 cells treated with E2 for various treatment durations and concentrations. D Typical images of microglia (Iba1-labeled) and M1 polarization markers (TNF-α) co-located by immunofluorescence. Cytotoxicity is detected by CCK-8 assays. HT22 cells (E) or primary neurons (F) are cultured either in E2 alone or co-cultured with microglia for 0–72 h. G Viability of primary neurons treated with E2 of various concentrations. H Typical images of neurons (NeuN-labeled) and COX1 co-located by immunofluorescence. Bar = 50 µm. I Quantification of the mean fluorescence intensity of COX1 per NeuN⁺ cell for (H). 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 or control groups as indicated by black asterisks or “ns” and to the OVX group as indicated by red asterisks. ns: no significant difference. Data are presented as mean ± the standard deviation (SD). Each experiment is repeated independently three. Data are based on a minimum of 10 animals in each group