Rishi Suresh

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 women^1,2. 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.

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