Journal: Journal of Neuroinflammation
Article Title: Estrogen receptor 1 activation suppresses microglia-derived Tnf-α signaling as a photoreceptor self-protection mechanism
doi: 10.1186/s12974-025-03669-z
Figure Lengend Snippet: Esr1 as an intrinsic self-protective mechanism. A GSEA of RNA-seq data from rd10 retinas at postnatal day 21 (P21) revealed significant positive enrichment of Estrogen-related pathways. B - C Immunofluorescence staining for Esr1 (green) demonstrated increased signal intensity in the entire retina of rd10 mice compared to congenic wt controls and negative staining controls. Esr1 was predominantly localized to IS, INL, and GCL. Negative control rd10 P21: n = 3; wt P21: 3; rd10 P21: 3. D - E Co-immunostaining of Iba1 (red) and Esr1 (green) further confirmed Esr1 expression in reactive microglia. Panel ( D ) shows 20 × confocal imaging, revealing widespread Iba1 microglia exhibiting overlapping Esr1 immunoreactivity. Panel ( E ) shows high-resolution 63 × imaging, demonstrating clear colocalization of Iba1 and Esr1 at the single-cell level, with both markers displaying shared positive signal within individual microglial somata and processes. F - G TUNEL staining (magenta) of ex vivo rd1 retinal explants showed that treatment with 10 µM E2, E2 + 10 nM Esr1 antagonist AZD9496, or 15 nM Esr1 agonist PPT markedly reduced ONL TUNEL positivity compared with untreated rd1 explants, consistent with the quantification in ( H ). Untr. wt: 3; Untr. rd1: 12; E2 rd1: 6; E2 + AZD9496 rd1: 4; PPT rd1: 4. H - I TUNEL staining and quantification of ex vivo rd10 retinal explants under identical treatment conditions revealed that E2 increased ONL TUNEL positivity, and E2 combined with the Esr1 antagonist AZD9496 further exacerbated photoreceptor cell death. In contrast, PPT treatment markedly reduced ONL cell death. J - K In vivo single intravitreal injection of PPT from P16-P23 significantly decreased ONL TUNEL positivity in rd10 mice. Untr. rd10: 13; PPT rd10: 13. L - M The same treatment improved dark-adapted ERG a-wave amplitudes, indicating partial functional rescue of photoreceptor responses. Scatter plots depict the percentage of positive cells in the ONL or the total fluorescence intensity; each dot corresponds to one independent retinal sample. Error bars: SD; significance levels: ns = p > 0.05; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. OS = outer segment, IS = inner segment, ONL = outer nuclear layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer, NFL = nerve fiber layer; scale bar = 50 µm. Dashed lines indicate untreated mutant or wt baseline levels. DAPI (grey) was used as nuclear counterstain
Article Snippet: After 48 h in culture, explants were treated with 10 μM E2 [ ] (HY-B0141; MedChemExpress, Sollentuna, Sweden), 10 μM E2 plus 10 nM AZD9496 [ ] (HY-12870; MedChemExpress), 15 nM PPT [ ] (HY-100689; MedChemExpress), 0.63 nM Etanercept [ ] (ETN, HY-108847; MedChemExpress), 6.7 μM Infliximab [ ] (IFX, HY-P9970; MedChemExpress), 65 μM R7050 [ ] (HY-110203; MedChemExpress), or 4 μM AZD8797 [ ] (HY-13848; MedChemExpress).
Techniques: RNA Sequencing, Immunofluorescence, Staining, Negative Staining, Negative Control, Immunostaining, Expressing, Imaging, TUNEL Assay, Ex Vivo, In Vivo, Injection, Functional Assay, Fluorescence, Mutagenesis
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