Neuroprotective effects of estrogens following ischemic stroke

Abstract

Our laboratory has investigated whether and how 17beta-estradiol (E(2)) protects the brain against neurodegeneration associated with cerebrovascular stroke. We have discovered that low, physiological concentrations of E(2), which are strikingly similar to low-basal circulating levels found in cycling mice, dramatically protect the brain against stroke injury, and consequently revealed multiple signaling pathways and key genes that mediate protective action of E(2). Here we will review the discoveries comprising our current understanding of neuroprotective actions of estrogens against ischemic stroke. These findings may carry far reaching implications for improving the quality of life in aging populations.

Fig. 1

Low, physiological levels of E₂ exert neuroprotection. E₂ protects against neuronal death in a stroke model in which the middle cerebral artery is permanently occluded. A: C57BL/6 J mice were ovariectomized and immediately implanted with capsules containing either oil or E₂ for 1 week. Subsequently, animals underwent experimental ischemia by middle cerebral artery occlusion and were killed 24 h after the onset of injury. Infarct volumes were measured using TTC staining in oil- or E₂-treated mice. B–D: E₂ treatment significantly reduced the total infarct volume (B, P < 0.02) and the extent of injury in both the cortex (C, P < 0.05) and striatum (D, P < 0.03). Data represent the mean ± SEM of 8–11 animals per group. Figure reprinted with permission from [85].

Fig. 2

E₂ attenuates markers of apoptosis. A, Photographs showing TUNEL-positive cells in the ischemic cortex from oil- and E₂-treated animals at 4, 8, and 24 h after stroke injury. B, E₂ significantly attenuates the number of TUNEL-positive cells during early (#P < 0.05) and late (*P < 0.05) phases of ischemic injury compared to oil-treated animals. Data represent the mean ± SEM of 8–10 animals per group. Figure reprinted with permission from [68].

Fig. 3

E₂ and iNOS display complementary neuroprotective interactions during MCAO. A, B: E₂ reduces infarct volume in the cortex and striatum of WT mice ( *, P < 0.05), but does not further suppress infarct size in iNOS−/− mice. iNOS−/− oil-treated mice were also protected during stroke compared to WT oil-treated mice (#, P < 0.05); n = 8–14 mice/treatment/genotype. C, D: NOS2 gene expression was significantly higher in WT oil-treated mice than in WT E-treated mice in the cortex and striatum (*P < 0.05). NOS2 gene expression was measured in 1-mm tissue micropunches adjacent to the tissue infarct in cortex and striatum of WT mice using qRT-PCR. n = 4–5 mouse cortex or striatum samples/hormone treatment. All values represent means ± SEM. Figure reprinted with permission from [9].

Fig. 4

E₂ suppresses peripheral cytokines following MCAO. MIP1α /CCL3 was significantly suppressed by E₂-treatment after MCAO injury in WT mice (*, P < 0.05), while E₂ provided no further suppression in iNOS−/− mice compared to oil-treated controls. As with infarct volume, an effect of genotype was observed in iNOS−/− oil-treated mice which were also protected during stroke compared to WT oil-treated mice (#, P < 0.05, n = 8–12 mice/genotype/treatment). Values represent mean ± SEM.

Fig. 5

E₂ increases synaptic density and size. A–F, Double-label ICC for NR1 (green) and vGlut1 (red); yellow in the superimposed images indicates colocalization. G–H, E₂ treatment significantly increased the density of colocalized NR1 and vGlut1 clusters and cluster size (48 h: P < 0.0005; 6 d: P < 0.003; Student’s t test). All values are mean ± SEM. Scale bars = 40 μm. Figure reprinted with permission from [37].

Fig. 6

E₂ significantly increases the number of BrdU⁺/Dcx⁺ newborn neurons. A, B: Confocal micrographs of BrdU⁺ cells (green) double-labeled with early neuronal marker doublecortin (Dcx, red) in the ipsilateral SVZ of oil- (A) vs. E₂- (B) treated mice at 96 h after MCAO injury. C: Higher magnification of panel B to demonstrate colocalization of BrdU⁺/Dcx⁺. Arrows indicate representative double-labeled cells. D: E₂ significantly increased the number of BrdU⁺/Dcx⁺ newborn neurons (*P = 0.0008, n = 6–7). E, F: BrdU⁺ cells (green) did not co-label with markers for mature neuron (NeuN, red; E) or astrocyte (GFAP, red; F). CC, corpus callosum; STR, striatum. Values represent mean ± SEM. Figure reprinted with permission from [85].

Fig. 7

E₂ attenuates ischemia-induced neuroinflammation. A, B: photomicrographs of mouse brain sections stained with a marker for activated microglia (lectin). Mice were ovariectomized and treated with oil or E₂ for 1 week prior to MCAO-induced ischemic injury, and collected at 24 h after injury. C: Ischemic injury increased the expression of IL-6 (*, P < 0.0001; oil-treated mice; *, P < 0.005; E₂-treated mice) on the injured (ipsilateral) side of the brain compared to the contralateral side. E₂ treatment attenuated ischemia-induced production of IL-6 (#P = 0.0271) on the ipsilateral side of the ischemic brain (n = 5–6 per experimental group). All values represent mean ± SEM. Figure reprinted with permission from [84].

Fig. 8

E₂ facilitates migration of newborn neurons. A, B: Confocal photomicrographs of Dcx⁺ cells migrating toward the ischemic boundary on the ipsilateral hemisphere from oil- (A) and E₂- (B) treated mice at 2 weeks after the onset of MCAO-induced injury.