Introduction. Ischemic stroke is a condition in which the brain is abruptly deprived of oxygen, leading to functional deficits. Epidemiologic studies have shown that young women experience strokes less frequently than similarly aged men; however, this trend reverses in post-menopausal women1,2. Based on these trends, researchers hypothesized that estrogen might be neuroprotective in ischemic stroke. However, the Women’s Health Initiative Study showed that estrogen or estrogen/progestin supplementation therapy in post-menopausal women increased the risk of stroke and breast cancer3. In studying the mechanisms responsible for this failed translation to the clinic, researchers have hypothesized that selectively stimulating estrogen receptor beta (ER-B) could lead to improved clinical outcomes in post-menopausal women with ischemic strokes. Methods. Hippocampal slices from Sprague-Dawley female rats 9-11 days old were treated with an ER inhibitor (ICI 182780), ER-a selective agonist (PPT), or an ER-B selective agonist (DPN). Propidium iodide (PI) staining was used to evaluate cell survival following oxygen glucose deprivation (OGD)4. ER-B was knocked down using an antisense oligodeoxynucleotide (missense oligonucleotide control) and the percentage of live neurons was recorded4. Primary astrocyte culture used hippocampal tissue from 1-day-old C57BL mice and was stained for Glial Fibrillary Acidic protein (GFAP)5. PPT, DPN treatments were performed with the astrocyte culture to study their effects on GFAP expression and apoptosis (via caspase-3 protein expression)5. Results. Hippocampal slices subjected to OGD and treated with DPN showed PI fluorescence values of 22+2 (p<0.05), while PPT showed values of 49+3 (p>0.05)4. Silencing of ER-B using an antisense oligodeoxynucleotide resulted in a live neuronal count of 150+9 (13% of control, p<0.001) while the missense condition resulted in a live neuronal count of 705+28 (64% of control)5. Astrocytes preferentially expressed ER-B relative to ER-a (p<0.05)5. DPN treatment caused a significant increase in GFAP protein expression relative to PPT treatment in cultured astrocytes (p<0.05)5. Western blot following DPN treatment showed decreased caspase-3 protein expression in response to ischemia relative to controls (p<0.05)5. Conclusions. Selective ER-B activation shows increased protection against cell death following oxygen glucose deprivation. Furthermore, silencing of ER-B appears to diminish estrogen’s neuroprotective effect in OGD. Astrocyte culture studies show that ER-B is expressed at a higher level than ER-a and that activation of ER-B via DPN increases GFAP expression. Finally, ER-B activation was shown to reduce neuronal apoptosis in OGD. These results suggest that selective ER-B activation might be responsible for mediating the neuroprotective role of estrogen.
- Koellhoffer EC, Mccullough LD. The Effects of Estrogen in Ischemic Stroke. Translational Stroke Research. 2013;4(4):390-401. doi:10.1007/s12975-012-0230-5.
- Girijala RL, Sohrabji F, Bush RL. Sex differences in stroke: Review of current knowledge and evidence. Vascular Medicine. 2016;22(2):135-145.
- Kaplan R, Salazar C, Wassertheil-Smoller S. Stroke Findings in the Womens Health Initiative. Seminars in Reproductive Medicine. 2014;32(06):438-446.
- Raval AP, Borges-Garcia R, Moreno WJ, Perez-Pinzon MA, Bramlett H. Periodic 17β-Estradiol Pretreatment Protects Rat Brain from Cerebral Ischemic Damage via Estrogen Receptor-β. PLoS ONE. 2013;8(4). doi:10.1371/journal.pone.0060716.
- Ma Y, Guo H, Zhang L, et al. Estrogen replacement therapy-induced neuroprotection against brain ischemia reperfusion injury involves the activation of astrocytes via estrogen receptor β. Scientific Reports. 2016;6(1). doi:10.1038/srep21467.