Table 1.
Material information for in vitro experiments.
Table 2.
primers used for in vitro PCR analyses.
Table 3.
material and medication for surgeries.
Table 4.
Material information for ex vivo PCR analyses.
Table 5.
Primers used for ex vivo PCR analyses.
Table 6.
primary antibodies for immunohistochemical staining Supplemental Information available zenodo; “S6 Tabl_Antibody_Registry_beta_ID_Tab6_Tab7”.
Table 7.
Secondary antibodies for immunohistochemical staining Supplemental Information available zenodo; “S6_Antibody_Registry_beta_ID_Tab6_Tab7”.
Fig 1.
Rat model of estradiol deficit.
A) Experimental Groups, created with BioRender.com B) Serum concentration of hormones depending on age (green = 6 months, red = 22 months). C) Serum concentration of hormones depending on surgical early menopause or during biological ageing (orange = Sham, red = OVX). Mann-Whitney tests were performed and the results are shown as boxes and whiskers (number of serum samples: 6Mo n = 6, 22Mo n = 12, Sham n = 6, OVX n = 6, *P < 0.05, **P < 0.01, ***P < 0.001).
Fig 2.
A) HE stained sagittal sections (400x magnification, scale bar = 100µm); left column shows the inner retina, right column shows the RPE. B) Cell loss per 100pixel GCL, C) Cell loss per 100pix2 ONL (green = 6 months, orange = 22 months Sham, red = 22 months OVX). Unpaired t-tests were performed and the results are shown as boxes and whiskers (n = 25 measuring points per group; 5 eyes from 5 different animals with 5 measuring points per eye, **P < 0.01, ****P < 0.0001). Raw data available zenodo; “Fig2_HE_samples_cell_counting”.
Fig 3.
Marker of cellular senescence.
IHC staining of p16 (green) and p21 (orange) in sagittal sections of rat retina (200x magnification, scale bar = 100µm). Raw data available zenodo; “Fig3_samples_p16p21_staining”.
Fig 4.
Structural changes in the RPE-layer.
A) Segmentation of phalloidin-stained RPE cells (200x magnification, scale bar = 50µm). Raw data available zenodo; “S2_raw_files_phalloidin_staining_Fig4” B) Segmentation from row A as violin plots. C) Changes in the morphology of the RPE-layer analyzed with the AI-supported software AIVIA (green = 6 months, orange = 22 months Sham, red = 22 months OVX). The raw data by AIVIA were compared using Mann-Whitney tests and the results are shown in graphs as mean ± SEM (6Mo; n = 58 scans out of 5 animals, 22Mo Sham; n = 46 scans out of 8 animals and 22Mo OVX; n = 80 scans out of 11 animals, ****P < 0.0001, ***P < 0.001).
Fig 5.
Estrogen receptors in the RPE.
A) Ethidium bromide gel of PCR amplicons of the receptor ERα and ERβ in ARPE-19 cells. Raw data available zenodo; “S1_raw_images_Fig5” B) Gene expression change depending on estrogen, in ARPE-19 cells (gray = ARPE-19 without estradiol; n = 28, blue = ARPE-19 + 4µM estradiol; n = 14), Mann-Whitney tests were performed and the results are presented in the diagram as mean ± SEM (***P < 0.001, ****P < 0.0001). C) qPCR detection of the receptors ERα and ERβ in rat RPE samples (green = 6 months; n = 6, light orange = 22 months naïve; n = 6, orange = 22 months Sham; n = 5, red = 22 months OVX; n = 5), analyzed by using t-tests and presented as boxes and whiskers (*P < 0.05). D) IHC staining of ERβ (orange), left in the inner retina (200x magnification, scale bar = 100µm), right in the RPE (400x magnification, scale bar = 50µm). Raw data available zenodo; “Fig5_samples_ERß_staining”.
Fig 6.
Estrogen impact on inflammatory processes in the RPE.
A) Gene expression changes of ARPE-19 cells under inflammatory (TNFα) conditions (grey = without estradiol; n = 14, blue = + 4µM estradiol; n = 7), analyzed by Mann-Whitney tests and presented in the diagram as mean ± SEM (*P < 0.05, **P < 0.01). B) Ccl2 expression level of the rat RPE depending on the estrous cycle (green = 6 months; n = 6, light orange = 22 months naïve; n = 6, orange = 22 months Sham; n = 5, red = 22 months OVX; n = 5), evaluated by Mann-Whitney tests and shown as boxes and whiskers (*P < 0.05). C) Images of the Iba1 analysis with the AI-supported software AIVIA (200x magnification, scale bar = 50µm). Raw data available zenodo; “S3_raw_files_Iba1_staining_Fig6”. D) AI-supported analysis of Iba1+ cells (n = 5 scans from 5 different animals per group). E) Counting of amoeboid Iba1+ cells (with scans from 5 animals per group with the following number of scans: 6 months; n = 53, 22 months Sham; n = 25, 22 months OVX; n = 36).