The goal of this study was to elucidate the mechanisms of 17β-estradiol (E2) antioxidant and neuroprotective actions in stroke. The results reveal a novel extranuclear receptor-mediated antioxidant mechanism for E2 during stroke, as well as a hypersensitivity of the CA3/CA4 region to ischemic injury after prolonged hypoestrogenicity. E2 neuroprotection was shown to involve a profound attenuation of NADPH oxidase activation and superoxide production in hippocampal CA1 pyramidal neurons after stroke, an effect mediated by extranuclear estrogen receptor α (ERα)-mediated nongenomic signaling, involving Akt activation and subsequent phosphorylation/inactivation of Rac1, a factor critical for activation of NOX2 NADPH oxidase. Intriguingly, E2 nongenomic signaling, antioxidant action, and neuroprotection in the CA1 region were lost after long-term E2 deprivation, and this loss was tissue specific because the uterus remained responsive to E2. Correspondingly, a remarkable loss of ERα, but not ERβ, was observed in the CA1 after long-term E2 deprivation, with no change observed in the uterus. As a whole, the study reveals a novel, membrane-mediated antioxidant mechanism in neurons by E2 provides support and mechanistic insights for a “critical period” of E2 replacement in the hippocampus and demonstrates a heretofore unknown hypersensitivity of the CA3/CA4 to ischemic injury after prolonged hypoestrogenicity.
The steroid hormone 17β-estradiol (E2) has been implicated to be neuroprotective in a variety of neurodegenerative disorders, such as stroke, Parkinson's disease, and Alzheimer's disease (Simpkins et al., 1997; Sherwin, 2003; Miller et al., 2005; Brann et al., 2007; Henderson, 2008; Morissette et al., 2008), although the mechanism for such broad-based neuroprotection remains unclear. With respect to stroke, E2 has been shown to be neuroprotective in rodent models of both focal and global cerebral ischemia (Simpkins et al., 1997; Toung et al., 1998; Miller et al., 2005; Brann et al., 2007; Zhang et al., 2008). Furthermore, it is well known that women are “protected” against stroke relative to men, at least until menopause (Roquer et al., 2003; Murphy et al., 2004; Niewada et al., 2005), and that after menopause, women reportedly have a worse stroke outcome compared with males (Di Carlo et al., 2003; Niewada et al., 2005). E2 has also been implicated to act in the hippocampus to enhance synaptic plasticity and cognitive function (Sandstrom and Williams, 2001; Li et al., 2004; Sherwin, 2007b; Spencer et al., 2008). Interestingly, long-term ovariectomy (surgical menopause) has been shown to be correlated with an increased risk of cognitive decline and dementia in humans (Rocca et al., 2007, 2008; Shuster et al., 2008).
In contrast to the beneficial effects reported for estrogen in animal and observational studies, the Women's Health Initiative (WHI) study failed to find a beneficial cardiovascular/neural effect of hormone replacement therapy (HRT) and in fact found an increased risk for stroke and dementia in postmenopausal women receiving HRT (Shumaker et al., 2003; Wassertheil-Smoller et al., 2003; Anderson et al., 2004; Espeland et al., 2004). However, it should be pointed out that the average age of subjects in the WHI study was 63–65 years, which is far past the menopause. This has led Sherwin and others (Maki, 2006; Sherwin, 2007a; Sherwin and Henry, 2008) to suggest that there exists a “critical period” for estrogen beneficial effect in the brain, in which estrogen replacement may need to be initiated at perimenopause to observe its beneficial effects on neuroprotection and cognition. In potential support of this hypothesis, rodent studies have shown that neuroprotection of the cerebral cortex by E2 is lost in long-term E2-deprived animals after middle cerebral artery occlusion (MCAO) (Suzuki et al., 2007).
Several important questions have arisen out of this body of work: (1) how does E2 exert a broad-based neuroprotective effect in different neurodegenerative disorders, including stroke, (2) is there a critical period for E2 protection of the hippocampus CA1 region, and (3) what is the mechanism underlying such a critical period and is it tissue specific? The current study sheds light on these important questions by demonstrating a novel, extranuclear receptor-mediated antioxidant mechanism of E2 in hippocampal CA1 neurons to suppress ischemic activation of NOX2 NADPH oxidase, a membrane enzyme that generates the highly reactive free radical, superoxide (O2−) (Bedard and Krause, 2007). NOX2 NADPH oxidase is highly localized in the hippocampal CA1 region (Serrano et al., 2003), and its activation is dependent on forming an active complex with several cytosolic factors (p47phox, p67phox, and p40phox) and activated Rac1, which translocate to the membrane after activation (Serrano et al., 2003; Bedard and Krause, 2007). The current study also demonstrates that a critical period exists for the antioxidant and neuroprotective effects of E2 in the hippocampus CA1 region, which are tissue specific, because the uterus remains sensitive to E2 after a period of prolonged hypoestrogenicity. Finally, the hippocampal CA3/CA4 region also showed a marked hypersensitivity to ischemic damage after prolonged hypoestrogenicity, which may explain the increased risk of cognitive decline and dementia observed in women after natural or surgical menopause.
Materials and Methods
Global cerebral ischemia.
Adult (3-month-old) Sprague Dawley female rats were bilaterally ovariectomized. Placebo (Pla) or E2 Alzet minipumps (0.025 mg; 14–21 d release) were implanted subcutaneously in the upper mid-back region under the skin at the time of ovariectomy [immediate (Imm)] and global cerebral ischemia (GCI) performed 1 week later. In some animals, long-term E2 deprivation was performed in which the animals were ovariectomized and, 10 weeks later (10W), placebo or E2 minipumps were implanted, and 1 week later GCI was performed (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). The dose of E2 used produces serum E2 levels of ∼10 pg/ml, which represents physiological low diestrus I levels of E2 (Zhang et al., 2008). For GCI, all animals (except sham control) underwent four-vessel occlusion GCI performed as described previously (Pulsinelli and Brierley, 1979; Pulsinelli and Buchan, 1988; Zhang et al., 2006a, 2008). Briefly, 24 h after electrocautery of the vertebral arteries, the common carotid arteries (CCAs) were occluded with aneurysm clips to induce 10 min forebrain ischemia. Animals that lost their righting reflex within 30 s and whose pupils were dilated and unresponsive to light during GCI were selected for the experiments, because this indicates successful GCI. The clips were then removed, and the blood flow through the arteries was confirmed before the wound was sutured. Rectal temperature was maintained at 36.5–37.5°C throughout the experiment with a thermal blanket. The animals of the sham group underwent identical procedures except that the CCAs were simply exposed but not occluded.
Histological examination of the ischemic brain was performed by neuronal-specific nuclear protein (NeuN) and Fluoro-Jade B as described previously by our laboratory (Zhang et al., 2008). Briefly, after perfusion with 0.9% saline followed by 4% paraformaldehyde (PFA) in 0.1 m phosphate buffer, the brains were postfixed, cryoprotected with 30% sucrose until they sank, and frozen sectioned (20 μm) in the coronal plane of the dorsal hippocampus (∼2.5–4.5 mm posterior from bregma). Every fifth section was collected and used for staining. Staining for NeuN and Fluoro-Jade B was performed using a mouse anti-NeuN monoclonal antibody (1:500; Millipore Bioscience Research Reagents) and Fluoro-Jade B (AG310; Millipore Bioscience Research Reagents) as described in detail previously by our laboratory (Zhang et al., 2008). Images were captured on an LSM510 Meta confocal laser microscope (Carl Zeiss) as described previously by our laboratory (Wakade et al., 2008). Cells that positively stained with NeuN and negatively stained with Fluoro-Jade B were identified as “surviving neurons”; in contrast, double-stained yellow-colored cells represent CA1 neurons undergoing degeneration.
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end (TUNEL) staining was performed on the free-floating coronal sections using the In Situ Cell Death Detection kit (Roche) following the instructions of the manufacturer. Briefly, after washing with 0.1% PBS–Triton X-100, the slides were permeabilized with 10 μg/ml proteinase K in 10 mm Tris/HCl, pH 7.4, for 15 min and incubated with TUNEL reaction mixture including enzyme solution (terminal deoxynucleotidyl transferase) and tetramethylrhodamine-labeled TUNEL-positive nucleotides in a humidified chamber for 1 h at 37°C. Slides for negative control were incubated with the label solution without terminal transferase for TUNEL. Samples were analyzed with a LSM510 Meta confocal microscope. For quantitative analyses, the number of surviving neurons and TUNEL-positive cells per 250 μm length of medial CA1 pyramidal cell layer were counted bilaterally in four to five sections per animal to provide a single value for each animal. A mean ± SE was calculated from the data in each group (n = 6–8 animals), and statistical analysis was performed as described below.
For DAB staining, sections were incubated with 10% normal goat/horse serum in PBS containing 0.1% Triton X-100 and 0.3% H2O2 for 1 h at room temperature to block nonspecific surfaces. Sections were then incubated with the primary antibodies overnight at 4°C in PBS containing 0.1% Triton X-100. The antibodies used were as follows: mouse anti-4-hydroxy-2-nonenal (4-HNE) (1:500; Genox), mouse anti-8-hydroxy-2′-deoxyguanosine (8-OHdG) (1:100; Genox), polyclonal rabbit anti-P47 (1:100; Cell Signaling Technology), and anti-NOX2/gp91phox (1:1000; Abcam). Afterward, sections were washed with the same buffer, followed by incubation with secondary biotinylated horse anti-mouse or goat anti-rabbit antibodies (Vector Laboratories) at a dilution of 1:200 in PBS containing 0.1% Triton X-100 for 1 h at room temperature. Sections were then washed, followed by incubation with ABC reagents for 1 h at room temperature in the same buffer. Sections were rinsed in the same buffer and incubated with DAB reagent according to the instructions of the manufacturer (Vector Laboratories) for 2–10 min. After DAB incubation, sections were washed briefly with distilled water and dehydrated in graded alcohols, cleared in xylene, and mounted using xylene-based mounting medium. Images were captured on an Axiophot-2 visible/fluorescence microscope using an AxioVision4Ac software system (Carl Zeiss).
Double/triple immunofluorescence staining.
Coronal sections were incubated with 10% normal donkey serum for 1 h at room temperature in PBS containing 0.1% Triton X-100, followed by incubation with appropriate primary antibodies overnight at 4°C in the same buffer. The following primary antibodies were used in different combinations: anti-NeuN (1:500; Millipore Bioscience Research Reagents), anti-phospho-H2A.X (1:100; Cell Signaling Technology), anti-p47phox, anti-GFAP, and anti-p-Akt (1:50; Santa Cruz Biotechnology), and anti-NOX2 (1:1000; Abcam). After primary antibody incubation, sections were washed for four times for 10 min at room temperature, followed by incubation with Alexa-Fluor594/647 donkey anti-mouse/rabbit, Alexa-Fluor488/594 donkey anti-rabbit/mouse, or Alexa-Fluor488/594 donkey anti-goat secondary antibody (1:500; Invitrogen) for 1 h at room temperature. Sections were then washed with PBS containing 0.1% Triton X-100 four times for 10 min, followed by three 5-min washes with PBS and briefly with water, and then mounted with water-based mounting medium containing anti-fading agents (Biomeda-Fisher Scientific). A simultaneous examination of negative controls (omission of primary antibody) confirmed the absence of nonspecific immunofluorescent staining, cross-immunostaining, or fluorescence bleed-through.
Confocal microscopy and image analysis.
All the double- and triple-labeled images were captured on an LSM510 Meta confocal laser microscope (Carl Zeiss) using either a 5× or 40× oil-immersion Neofluor objective (1.3 numerical aperture) with the image size set at 1024 × 1024 pixels. The following excitation/emission laser filter settings were used for various chromophores: an argon/2 laser was used for Alexa-Fluor488, with excitation maximum at 490 nm and emission in the range of 505–530 nm, a helium–neon laser was used for Alexa-Fluor594 with excitation maximum at 543 nm and emission in the range of 568–615 nm, and a second helium–neon laser was used for Alexa-Fluor647 with excitation maximum at 633 nm and emission in the range of 650–800 nm. The captured images were viewed and analyzed using LSM510 Meta imaging software.
In situ detection of superoxide production.
The production of O2− radicals was investigated using hydroethidine (HEt) (Invitrogen) as described previously by our group and others (Bindokas et al., 1996; Wakade et al., 2008). In the present study, HEt (1 mg/ml in 200 μl of PBS) was administered intravenously 30 min before ischemia. Animals were anesthetized using isoflurane 3 h after ischemia and transcardially perfused with cold PBS and 4% PFA. Sham non-ischemic control animals were also treated with HEt solution as O2− production control. Fluorescent intensity of the oxidized HEt was measured on a confocal laser microscope using an excitation wavelength of 543 nm, and the emission was recorded at wavelengths between 560 and 590 nm. The images were examined using LSM 510 image software, and the optical intensity of HEt signals in each recorded medial CA1 objective field was measured with the NIH Image J analysis software (version 1.30v). The mean intensity was determined from four fields in each animal, and values (four or five rats in each group) were expressed as fold changes versus sham control.
Brain homogenates and subcellular fractionations.
For brain tissue preparation, rats were killed under anesthesia at the indicated time points. Whole brains were removed, and the hippocampal CA1 and CA3/dentate gyrus (DG) regions were microdissected from both sides of the hippocampal fissure and immediately frozen in liquid nitrogen. Total protein, nuclear, cytosol, and crude membrane fractions were extracted as follows. Briefly, tissues samples were gently homogenized using a glass homogenizer in 1.2 ml of ice-cold buffer A containing 1.5 ml of 10 mm HEPES, pH 7.4, 0.5 mm MgCl2, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 50 mm NaF, 5 mm dithiotheitol, 10 mm β-phosphoglycerol, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride (PMSF), 1 mm 4-nitrophenyl phosphate, and protease inhibitor mixture (Sigma). Two hundred microliters of the above homogenates were separated and sonicated as total protein lysates. The remaining homogenates were centrifuged at 1000 × g at 4°C for 10 min to get supernatant (S1) fractions and nuclear fractions (P1). P1 fractions were then extracted with our previously described procedure (Zhang et al., 2008) and used as a crude nuclear fraction. S1 fractions were centrifuged at 13,000 × g for 20 min at 4°C (results in S2 fractions and P2 mitochondria/microsome fractions) and further centrifuged at 100,000 × g for 30 min to obtain the cytosolic fractions (S3) and the plasma membrane-enriched fractions (P3). The pellet membranes fractions were resuspended in buffer A containing 0.1% Triton X-100 for 10 s by sonication. The protein concentrations were determined by the Modified Lowry Protein Assay (Pierce). All the samples were stored at −80°C until use.
Immunoprecipitation and Western blotting.
For immunoprecipitation (IP), the supernatant (S2) fractions (each containing 400 μg of protein) were diluted fourfold with HEPES buffer containing 50 mm HEPES, pH7.4, 150 mm NaCl, 10% glycerol, 1% Triton X-100, and 1 mm each of EGTA, EDTA, PMSF, and Na3VO4. Samples were preincubated for 1 h with 20 μl of protein A/G and then centrifuged to remove any protein adhered nonspecifically to the protein A/G. The supernatant was incubated with 5 μg of proper antibodies for 4 h at 4°C. After the addition of protein A/G-Sepharose, the mixture was incubated at 4°C for an additional 2 h. Samples were washed three times with HEPES buffer and eluted by SDS-PAGE loading buffer and then boiled for 5 min.
Western blotting was performed as described in detail previously (Zhang et al., 2008). The antibodies used were as follows: phospho-H2A.X (1:1000; Cell Signaling Technology), β-actin (1:2000; Sigma), Na+/K+-ATPase (1:1000; Millipore Bioscience Research Reagents), Rac1 (1:1000; BD Biosciences Transduction Laboratories), NOX2 (1:1000; Abcam), phospho-Rac1 (Ser71) (1:1000; Cell Signaling Technology); antibodies to pan-cadherin, p47phox, p67phox, GFAP, p-Akt, Akt, phosphatidylinositol 3-kinase (PI3K)–p85, ERα, and ERβ for IP or Western blots (1:200) were from Santa Cruz Biotechnology. The membrane was then washed with TBS containing Tween 20 to remove unbound antibody, followed by incubation with Alexa-Fluor 680 goat anti-rabbit/mouse IgG for 1–2 h at room temperature. Bound proteins were visualized using the Odyssey Imaging System (LI-COR Bioscience), and semiquantitative analysis of the bands was performed with NIH Image J analysis software (version 1.30v). Band densities for the indicated proteins were normalized and expressed relative to total proteins, actin, or cadherin as indicated in the figures. Normalized means were then expressed as fold changes of the corresponding value for control (sham-operated) animals. A mean ± SE was calculated from the data from all the animals for graphical presentation and statistical comparison.
NADPH oxidase activity and superoxide production assay.
NADPH oxidase activity was determined based on superoxide-induced lucigenin photoemissions. For assaying NADPH oxidase enzymatic activity, 50 μg of membrane fractions were used. Enzyme assays were performed in a final volume of 1 ml containing 50 mm Krebs'–Ringer's phosphate buffer, pH 7.0, 1 mm EGTA, 150 mm sucrose, 0.5 mm lucigenin, 0.1 mm NADPH, and tissue homogenate. Enzyme reactions were initiated with the addition of NADPH. No enzymatic activity could be detected in the absence of NADPH. Photoemissions, expressed in terms of relative light units (RLU), were measured every min for 5 min using a luminometer. Assays were performed in the dark at room temperature with appropriate controls. The rate of NADPH consumption was monitored by measuring the means values in absorbance (340 nm), and NADPH oxidase activity was normalized by the amount of protein and the change in optical density (OD). Activity was calculated as OD per micrograms of protein per minute.
Superoxide production was measured from the indicated supernatant (S2) fractions using a LumiMax Superoxide Anion Detection kit (Stratagene) following the protocol of the manufacturer. Briefly, 50 μg of sample proteins were suspended in 100 μl of superoxide anion (SOA) assay medium and then mixed with 100 μl of reagent mixture containing 0.2 mm luminal and 0.25 mm enhancer in SOA assay medium. Light emissions at 30 s intervals were recorded by a standard luminometer. Values were standardized to the amount of protein, and photons of light counted were expressed as RLU per micrograms of protein. A mean ± SE were calculated from the data collected in each group for graphical depiction expressed as fold changes versus sham control group. Statistically analyses of the data were performed as described below.
Rac1–GTP binding assay.
Rac1 activation assays were performed using PAK1–PBD color agarose beads (Cell Biolabs). Briefly, 400 μg samples were mixed with 20 μl of PAK1–PBD agarose beads and incubated for 1 h at 4°C. The reaction was terminated by addition of MgCl2. The agarose beads were collected by spinning at 12,000 × g for 1 min at 4°C, and the supernatants were removed. Precipitated complexes were washed three times with magnesium-containing TBS buffer and boiled in sample buffer. Proteins were separated by 10% SDS-PAGE, transferred onto nitrocellulose membrane, and detected by immunoblotting using an anti-Rac1-specific antibody.
Administration of drugs.
The following compounds were bilaterally infused into the lateral ventricles (from bregma: anteroposterior, ±0.8 mm; lateral, 1.5 mm; depth, 3.5 mm) 30 min before induction of GCI: the estrogen receptor antagonist ICI182,780 (7α,17β-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol) [50 μg (Tocris Bioscience), dissolved in 5 μl of 50% DMSO], gp91ds–tat, and its scrambled control peptide (Scr) (100 ng each, 5 μl in saline; synthesized by AnaSpec). The Rac1 inhibitor NSC23766 (6-N-[2-[5-(diethylamino) pentan-2-ylamino]-6-methylpyrimidin-4-yl]-2-methylquinoline-4,6- diamine chloride) (25 μl in 5 μl of sterile saline), 17β-estradiol dendrimer conjugate (EDC) (10 μm in 5 μl of saline), and the phosphatidylinositol ether lipid analog SH-5 ([(2R)-2-methoxy-3-octadecoxypropyl] (2,3,4-trihydroxy-6-methoxycyclohexyl) hydrogen phosphate) (25 μg in 10% DMSO), a recently discovered Akt-specific inhibitor (Alexis Biochemicals).
To investigate the roles of ERα and ERβ in E2 neuroprotection, 10 nmol of end-phosphorothioated HPLC-purified antisense oligodeoxynucleotides (AS-ODNs) mixed with 5 μl of in vivo-jetPEI (Polyplus Transfection) were administrated by bilateral cerebroventricular infusion every 24 h for 4 d before GCI. The last infusion was administered 30 min before CCA occlusion. The sequences used in this study were 5′-CATGGTCATGGTCAG-3′ for ERα AS-ODNs and 5′-GAATGTCATAGCTGA-3′ for ERβ AS-ODNs (from Integrated DNA Technologies). The same dose of scrambled missenses (MSs) (5′-ATCGTGGATCGTGAC-3′) was used as control. These ER antisense sequences have been well described in vivo previously by others (Liang et al., 2002; Edinger and Frye, 2007; Walf et al., 2008).
For intracerebroventricular injections, anesthetized rats were placed on a stereotaxic instrument. All drug infusions as listed above were performed using a Hamilton microsyringe at a flow rate of 1 μl/min. After injection, the needle was left in situ for 5 min before the complete 2 min retraction.
Statistical analysis was performed using either one-way or two-way ANOVA analysis, followed by Student–Newman–Keuls post hoc tests to determine group differences. When groups were compared with a control group (e.g., sham), Dunnett's test was adopted for post hoc analyses after ANOVA. When only two groups were compared, a Student's t test was used. Statistical significance was accepted at the 95% confidence level (p < 0.05). Data were expressed as mean ± SE.
E2 protects the hippocampus CA1 region from GCI-induced delayed neuronal cell death
Figure 1, A and B, shows the neuroprotective effect of E2 on the hippocampal CA1 region after GCI. As shown in Figure 1A, staining for NeuN (a neuronal marker) and Fluoro-Jade B (a neuronal degeneration marker) revealed that GCI (Pla) induced a profound loss of NeuN staining with an elevation of Fluoro-Jade B staining in the hippocampus CA1 region at 7 d after GCI reperfusion compared with sham control. Figure 1B shows quantification of number of “surviving” neurons (cells positive for NeuN but negative for Fluoro-Jade B) in the CA1 region from all animals, which confirms that E2 exerts a robust neuroprotective effect against cerebral ischemia. Additionally, staining for TUNEL, an apoptotic marker, revealed that GCI (Pla) significantly increased TUNEL staining and the number of TUNEL-positive cells in the CA1 region compared with sham control, with E2 significantly attenuating this effect (Fig. 1A,B). Furthermore, E2 neuroprotection appeared to be mediated by ERs because intracerebroventricular administration of the ER antagonist ICI182,780 reversed E2 effects on NeuN and Fluoro-Jade B staining (Fig. 1A), number of surviving neurons (Fig. 1B), and number of TUNEL-positive cells in the CA1 region (Fig. 1A,B).
E2 profoundly attenuates neuronal NADPH oxidase activation, superoxide anion (O2−) production, and oxidative damage in the hippocampal CA1 region after GCI
Because reactive oxygen species (ROS) can play a major role in damaging neurons after GCI reperfusion, we next examined whether E2 exerts an antioxidant effect through regulation of NADPH oxidase activation and O2− production in the hippocampal CA1 region at different times after GCI. As shown in Figure 2, A and B, NADPH oxidase activity and O2− production in the CA1 were significantly elevated in Pla versus sham control as early as 30 min after reperfusion, with peak NADPH oxidase activity and O2− levels observed at 3 h (approximately sixfold to sevenfold increase vs sham), followed by a sharp fall from 6 to 24 h. Intriguingly, low-dose E2 replacement profoundly attenuated NADPH oxidase activation and O2− production in the hippocampal CA1 region after reperfusion (Fig. 2A), an effect blocked by the ER antagonist ICI182,780 (Fig. 2B). NADPH oxidase activation and O2− production was not significantly elevated in the relatively resistant CA3/DG region after reperfusion, and E2 had no significant effect on NADPH oxidase activation and O2− production in the CA3/DG (supplemental Fig. 2D, available at www.jneurosci.org as supplemental material). O2− production was also assessed using the in situ oxidized HEt method, in which HEt, a marker of O2− production, is selectively taken up by cells and oxidized by O2− into ethidium, which provides a red fluorescence signal. As shown in Figure 2C, assessment of oxidized HEt signal in the CA1 region at 3 h after reperfusion revealed a robust induction of O2− in the Pla group compared with sham controls. E2 markedly attenuated the induction of O2− in the CA1, an effect blocked by the ER antagonist ICI182,780. In contrast to the CA1 region, the CA3/DG region showed low oxidized HEt signal at 3 h after reperfusion, which is in agreement with a relative lack of NADPH oxidase activation observed in the CA3/DG (supplemental Fig. 2C, available at www.jneurosci.org as supplemental material). Examination of oxidative damage markers revealed that, in agreement with reduction of NADPH oxidase activity and O2− by E2 after GCI, E2 markedly attenuated oxidative damage in the CA1 region at 24 h after GCI as measured by immunostaining for 4-HNE, a marker of lipid oxidative damage, as well as 8-OHdG, a marker of DNA oxidative damage (Fig. 2D). Additionally, 48 h after reperfusion, the Pla group displayed a dramatic increase in the CA1 region of phosphorylation of the histone protein H2AXser139, another well known marker of DNA damage in cells (Fig. 2Ea,Eb), and this effect was almost completely prevented by E2 treatment.
NOX2 is highly expressed in hippocampal CA1 neurons and inhibition of NADPH oxidase activation attenuates O2− production and neuronal cell death after GCI
The NOX2 isoform is a major isoform of NADPH oxidase and has been shown to be highly expressed in many brain regions, including the hippocampus (Serrano et al., 2003). We thus examined whether NOX2 is localized in neurons or astrocytes in the hippocampal CA1 region after GCI. Triple immunostaining in the CA1 region at 3 h after GCI in Pla animals for the neuronal marker NeuN, NOX2, and the glia marker GFAP revealed that NOX2 is predominantly localized in neurons and is found in the membrane and cytoplasmic compartments (Fig. 3A). p47phox, which forms a complex with NOX2 leading to NADPH oxidase activation, was shown to also exhibit a predominant neuronal localization. Examination of the CA3/DG region revealed that it possessed extremely weak immunostaining for NOX2 and p47phox compared with the CA1 region, potentially explaining its relative resistance to ischemic damage (supplemental Fig. 2A,B, available at www.jneurosci.org as supplemental material). To determine whether NADPH oxidase plays a major role in O2− production and oxidative damage in the CA1 after GCI, we used the NADPH oxidase competitive antagonist gp91ds–tat, which is a 9 aa peptide sequence of the p47Phox docking site on NOX2 and prevents p47phox from forming a complex with NOX2 (Rey et al., 2001). Figure 3B shows that gp91ds–tat administration abolished the enhanced membrane localization of p47phox in Pla animals observed at 3 h after reperfusion. In contrast, Scr had no effect on membrane p47phox levels. Co-IP studies revealed that NOX2–p47phox complex formation was markedly increased in Pla animals and Scr-treated controls compared with sham controls and that gp91ds–tat treatment significantly attenuated the NOX2–p47phox complex formation, thus demonstrating its effectiveness in blocking p47phox–NOX2 complex formation. We next examined gp91ds–tat effect on NADPH oxidase activation and O2− production after GCI. As shown in Figure 3C, gp91ds–tat, but not Scr, markedly attenuated the enhanced NADPH oxidase activation and O2− production in the CA1 region at 3 h after reperfusion. gp91ds–tat also significantly increased the number of surviving neurons in the CA1 region at 7 d after GCI compared with Pla or the scrambled peptide control-treated animals (Fig. 3D), suggesting that activation of neuronal NADPH oxidase and enhanced O2− production plays an important role in inducing neuronal death in the hippocampal CA1 region after GCI.
Membrane NADPH oxidase “complex formation” increases in hippocampal CA1 region after GCI and is attenuated by E2 treatment
Additional studies further demonstrated that membrane levels of the NOX2 activation factors p47phox, p67phox, and Rac1 show a stepwise increase after reperfusion with peak levels observed at 3 h after reperfusion with levels decreasing at 6 h and returning to close to sham levels by 24 h after reperfusion (supplemental Fig. 3, available at www.jneurosci.org as supplemental material). Total p47phox, p67phox, and Rac1 levels were not significantly changed at any time point after GCI reperfusion, suggesting that expression of the factors did not change after ischemia. As shown in Figure 4A–C, co-IP studies showed that complex formation of p47phox and p67phox with NOX2 is markedly increased in Pla animals compared with sham controls and that E2 significantly attenuates NOX2 complex formation with p47phox and p67phox. In agreement with these results, Western blot analysis of membrane fractions demonstrated that E2 significantly attenuated membrane translocation of p47phox and p67phox at 3 h after reperfusion but had no significant effect on total p47phox and p67phox levels (Fig. 4D,E).
Rac1 activation is critical for NADPH oxidase activation after GCI and is significantly attenuated by E2
We next examined the activation and role of Rac1 in NADPH oxidase activation, O2− production, and ischemic neuronal death in the CA1 region after GCI. E2 regulation of Rac1 activation and complex formation with NOX2 was also examined. Administration of the Rac1 inhibitor NSC23766 (Rac1-I) intracerebroventricularly 30 min before GCI markedly attenuated NADPH oxidase activation and O2− production in the CA1 region at 3 h after reperfusion (NADPH oxidase activity: vehicle, 7.70 ± 1.32-fold vs NSC23766, 3.32 ± 1.25-fold increase vs sham, p < 0.05; superoxide levels: vehicle, 6.21 ± 0.88-fold vs NSC23766, 2.14 ± 0.50 fold increase vs sham, p < 0.05). Furthermore, as shown in Figure 5A, the Rac1 inhibitor was neuroprotective as demonstrated by an increased number of surviving neurons in the CA1 compared with a vehicle control (vehicle, 7.60 ± 1.50 vs NSC23766, 38.20 ± 8.27 surviving neurons, p < 0.05). Rac1–GTP binding, which is critical for Rac1 activation, was also shown to be significantly elevated in Pla animals at 3 h after reperfusion compared with sham controls, and E2 attenuated this effect, suggesting that Rac1 activation is increased after GCI and that E2 attenuates the activation (Fig. 5B). In contrast, total Rac1 protein levels did not change significantly in any group. However, membrane levels of Rac1 and NOX2–Rac1 complex formation in the hippocampus CA1 were increased in the Pla group animals at 3 h after reperfusion compared with sham controls, and E2 treatment prevented this effect (Fig. 5C,D).
Extranuclear estrogen receptor-induced Akt signaling mediates E2 attenuation of NADPH oxidase activation, O2− production, and E2 neuroprotection after GCI
To determine the potential role of extranuclear estrogen receptors and nongenomic signaling in E2 antioxidant and neuroprotective actions, we used EDC, an E2 conjugate that cannot enter the nuclei of cells and thus is capable of exerting nongenomic but not genomic signaling (Harrington et al., 2006). We first intracerebroventricularly injected FITC-tagged EDC (FITC–EDC) (10 μm) to determine its uptake in the hippocampal CA1 region and its subcellular distribution at 3 h after reperfusion. As shown in supplemental Figure 4 (available at www.jneurosci.org as supplemental material), FITC–EDC signal was strongly localized in the CA1 region and less so in CA3 and DG regions. Additionally, FITC–EDC signal was extranuclear, localized in the membrane and cytoplasm of cells, confirming previous reports (Harrington et al., 2006). As shown in Figure 6A, EDC administration significantly enhanced p-Akt levels in the CA1 region at 3 h after reperfusion, and the effect appeared to involve ER mediation because it was blocked by ICI182,780. In other studies by our group, the enhanced activation of Akt by EDC was observed as early as 10 min after GCI, and peak levels were maintained out to 3 h (data not shown). E2 also enhanced p-Akt levels, and this effect was blocked by the Akt inhibitor SH-5 (Akt-I, intracerebroventricularly) (Fig. 6A). Confocal analysis of NeuN, p-Akt, and oxidized HEt staining of hippocampal sections confirmed EDC and E2 effect on enhancing p-Akt and extended the findings by demonstrating that the effect occurred in neurons (colocalized with NeuN) and that cells that had enhanced p-Akt levels had correspondingly low O2− production, as evidenced by low oxidized HEt staining and vice versa (Fig. 6B). Furthermore, the Akt inhibitor SH-5 reversed E2 attenuation of O2− levels, confirming that Akt signaling is critical for E2 suppression of O2− production. EDC also significantly attenuated the elevation of NADPH oxidase activity and O2− levels in the CA1 region after reperfusion, an effect blocked by ICI182,780 (Fig. 6C). Intriguingly, pretreatment with the Akt inhibitor SH-5 blocked the E2 suppressive effects on NADPH oxidase activation and O2− production in the CA1 region, demonstrating a critical role for Akt in mediating E2 antioxidant effects (Fig. 6C). EDC neuroprotection was also shown to involve Akt signaling because pretreatment with SH-5 significantly attenuated its neuroprotective effects in the CA1 region, as determined by NeuN and Fluoro-Jade B staining results (Fig. 6D). Finally, previous work by our group and others provided evidence that Akt can phosphorylate Rac1 at Ser71 and that this effect is correlated with reduced Rac1 activation (Kwon et al., 2000; Zhang et al., 2006b). We thus examined the effect of EDC and E2 on Rac1 phosphorylation and Rac1–GTP binding after GCI and determined the potential mediatory role of Akt. As shown in Figure 6E, both EDC and E2 enhanced phosphorylation of Rac1 at Ser71 in the CA1 region at 3 h after reperfusion, and their effect was correlated with a significant reduction in Rac1 activation. Furthermore, administration of the Akt inhibitor SH-5 markedly attenuated the ability of E2 to enhance phosphorylation of Rac1 and reduce Rac1 activation after GCI.
Evidence that ERα mediates E2 effects to attenuate NADPH oxidase activation, O2− production, and exert neuroprotection in the hippocampus CA1 after GCI
We next sought to determine whether the antioxidant and neuroprotective effects of E2 against cerebral ischemia were mediated by ERα or ERβ. To determine the specific role of ERα and/or ERβ, we used AS-ODNs to ERα and ERβ for knockdown of each receptor in the hippocampus. To determine that the ODNs reach the hippocampus after intracerebroventricular injection, we used Alexa488-labeled MS-ODNs (Alexa488N-MS). As shown in Figure 7A, 3 h after intracerebroventricular injection of Alexa488N-MS, the fluorescent Alexa488N-MS signal was predominantly localized in the hippocampal CA1 region. Additionally, double staining for NeuN revealed that the fluorescent Alexa488N-MS signal was located predominantly in the cytoplasm of neurons in the CA1 region. We next confirmed the efficacy of the ERα and ERβ AS-ODNs in knocking down ERα and ERβ in the hippocampal CA1 region after GCI reperfusion. As shown in Figure 7B, the ERα-AS-ODNs and ERβ-AS-ODNs induced a marked decrease in ERα and ERβ protein levels, respectively. MS-ODNs had no effect on ERα or ERβ protein levels, indicating that AS-ODNs effects were specific. As shown in Figure 7C, ERα-AS-ODNs but not ERβ-AS-ODNs significantly reversed the suppressive effect of E2 on NADPH oxidase activation and O2− production in the hippocampal CA1 region at 3 h after reperfusion, whereas MS-ODNs were without effect. Finally, Figure 7D shows that that ERα-AS-ODNs, but not ERβ-AS-ODNs, significantly attenuated the neuroprotective effect of E2 against GCI, suggesting that ERα mediates the neuroprotective actions of E2 in the hippocampus CA1. MS control oligos had no significant effect, indicating the specificity of the ERα-AS effect.
Long-term E2 deprivation leads to loss of E2 nongenomic signaling, antioxidant, and neuroprotective effects in the hippocampus CA1 after GCI, whereas E2 uterotropic effects are preserved
It has been proposed that the lack of beneficial cardiovascular and cognitive effect of E2 in the WHI studies could be attributable to estrogen replacement therapy being initiated in subjects too far past menopause, e.g., after too long a period of E2 deprivation. We thus examined whether a period of long-term E2 deprivation (10 weeks) would lead to diminishment of E2 signaling and actions in the hippocampal CA1 region after GCI. Additionally, the tropic effect of E2 on the uterus was examined to determine whether changes in E2 sensitivity after long-term E2 deprivation were brain specific. As shown in Figure 8A, the ability of E2 to exert neuroprotection in the hippocampal CA1 region is lost in long-term E2-deprived animals, as determined by NeuN and Fluoro-Jade B staining and counting of number of surviving neurons. Additionally, the ability of E2 to suppress NADPH oxidase activation and O2− production in the CA1 region at 3 h after reperfusion was lost in long-term E2-deprived animals (NADPH oxidase activity: Pla, 7.20 ± 0.65-fold vs E2, 6.83 ± 0.88-fold increase vs sham, no significant difference; superoxide levels: Pla, 5.81 ± 0.56-fold vs E2, 5.28 ± 0.96-fold increase vs sham, no significant difference). The ability of E2 to enhance Akt phosphorylation in the CA1 region at 3 h after reperfusion was also lost in long-term E2-deprived animals (Fig. 8B). Previous work has shown that ERα can interact with the p85 regulator subunit of PI3K, an effect enhanced by E2 treatment and critical for its ability to enhance phosphorylation of Akt (Simoncini et al., 2000). We thus performed co-IP studies for ERα and p85 from hippocampal CA1 samples at 3 h after GCI in immediate E2-treated (Imm) ovariectomized animals and in long-term E2-deprived animals (10W). As shown in Figure 8B, E2 enhanced ERα and p85 subunit interaction in the hippocampus CA1 in Imm animals, but this ability was lost in long-term E2-deprived animals (10W). Additionally, examination of NeuN and Fluoro-Jade B staining and counting of the number of surviving neurons in the CA1 revealed that the neuroprotective effect of EDC is lost in 10W animals (Fig. 8C) (vehicle, 8.25 ± 4.10 vs EDC, 12.26 ± 6.12 surviving neurons, no significant difference), as was its ability to enhance Akt phosphorylation (Fig. 8D) However, in contrast to the loss of E2 sensitivity observed in the hippocampus CA1 of 10W animals, the well known uterotrophic effect of E2 on the uterus was not lost in 10W animals, indicating that loss of E2 sensitivity after long-term E2 deprivation is tissue-specific (Fig. 8E).
ERα, but not ERβ, protein levels are significantly reduced in the hippocampal CA1 region after long-term E2 deprivation
Because E2 signaling and actions in the hippocampus were attenuated in long-term E2-deprived animals but its uterotrophic action in the uterus was preserved, we examined the hippocampal CA1 region and uterus for alterations in ERα and ERβ after long-term E2 deprivation. As shown in Figure 9A, Western blot analysis revealed a dramatic attenuation of ERα, but not ERβ, protein levels in the hippocampal CA1 region of 10W animals compared with Imm animals. Note that the reduction in ERα protein levels occurred in all groups, including sham controls, suggesting that long-term E2 deprivation leads to lower ERα levels regardless of treatment and that E2 and ischemia cannot reverse the suppression of ERα protein levels. Figure 9B shows double immunohistochemistry for NeuN and ERα in the CA1 region in Imm and 10W animals, which further confirms that ERα expression in hippocampal CA1 neurons is strongly attenuated in 10W animals compared with Imm animals; note the marked attenuation of “green” ERα signal and “yellow” ERα–NeuN merged signal in 10W animals. In contrast, examination of the uterus revealed no significant change of ERα or ERβ protein levels in 10W animals compared with Imm animals (Fig. 9C). Furthermore, E2 exerted a well characterized rapid downregulation of ERα and ERβ in the uterus of Imm animals consistent with other reports in the literature (Medlock et al., 1991; Nephew et al., 2000; Pillai et al., 2002; Kang et al., 2003), and this effect was preserved in 10W animals. This finding further confirms that E2 sensitivity and ERα/β protein levels are not attenuated in the uterus after a long-term period of E2 deprivation.
Long-term E2 deprivation leads to hypersensitivity of the hippocampal CA3/CA4 region to ischemic neuronal cell death
Additional examination of long-term E2-deprived animals revealed that prolonged E2 deprivation leads to hypersensitivity of hippocampal CA3/CA4 neurons to ischemic cell death after GCI. As shown in Figure 10A, sham animals from Imm or 10W groups show no damage or cell loss in any hippocampal region, as assessed by NeuN staining. In contrast, the Imm placebo-treated (Pla) group displayed a dramatic loss of neurons in the CA1 region but not CA3/CA4 or DG region of the hippocampus at 7 d after 10 min GCI. Of significant interest, 10W Pla animals displayed a dramatic loss of cells not only in the CA1 region but also in the CA3/CA4 region after 10 min GCI, suggesting an enhanced sensitivity of the CA3/CA4 to ischemic damage after long-term E2 deprivation (Fig. 10A). Figure 10B shows that E2 treatment of Imm animals profoundly protects the CA1 region from ischemic damage as expected. It also shows that ERα is highly expressed in the surviving cells in the CA1 region and also CA3 region. In contrast, Figure 10Bb reveals that E2 treatment initiated 10 weeks after ovariectomy does not protect the CA1 region and is unable to protect the CA3/CA4 region from ischemic damage. However, it is noteworthy that the few surviving cells in the CA1 and CA3 region at 7 d after reperfusion strongly express ERα (Fig. 10B, yellow merged cells).
It is well known that ischemic reperfusion can induce significant damage to the brain attributable to the enhanced generation of ROS, such as the highly reactive O2− ion (Chan, 1996; Peters et al., 1998; Sugawara et al., 2005; Muranyi and Li, 2006; Brann et al., 2007), which can give rise to other damaging ROS such as hydroxyl ion and peroxynitrite (Mattson et al., 2000). The current study provides evidence that the membrane enzyme NADPH oxidase plays a major role in the generation of O2− in the hippocampal CA1 region after ischemic reperfusion and that inhibition of NADPH oxidase activation by either a competitive NADPH oxidase inhibitor (gp91ds–tat) or E2 treatment profoundly protects the hippocampal CA1 region from oxidative damage and neuronal cell death. The source of NADPH oxidase activity and O2− generation after ischemic reperfusion appears to be primarily neuronal, because enhancement of oxidized HEt, an in situ marker of O2− production, occurred predominantly in neurons. Furthermore, both NOX2 and p47phox were highly colocalized in neurons but not in glia after GCI.
Our study suggests that the NOX2 isoform of NADPH oxidase has a critical role in the enhanced NADPH oxidase activation and O2− production after GCI, because NOX2 and p47phox were highly colocalized in CA1 neurons, and NOX2–phox complex formation increased significantly at 3 h after GCI reperfusion, the time of peak NADPH oxidase activity and O2− production. Furthermore, administration of the competitive NOX2 NADPH oxidase inhibitor gp9lds–tat dramatically attenuated NADPH oxidase activation and O2− production after reperfusion and strongly attenuated neuronal cell death. NOX2 activation is also known to require activated Rac1 (Bedard and Krause, 2007), and our study showed that a Rac1 inhibitor strongly reduced NADPH oxidase activation and O2− production after GCI, further implicating NOX2. Additional work by others suggests that NOX2 also has a role in oxidative damage in the cerebral cortex, because cortical infarct damage was reduced after MCAO in NOX2 knock-out mice or after administration of the NADPH oxidase inhibitor apocynin (Kahles et al., 2007; Tang et al., 2008; Chen et al., 2009). Thus, NOX2 NADPH oxidase appears to play a major role in ischemic ROS generation and oxidative damage in the brain. It is important to note that, in our study, the hippocampal CA3/DG region, in contrast to the CA1 region, showed very low expression of NOX2 and phox subunits and did not exhibit a significant elevation of NADPH oxidase activity or O2− production after GCI reperfusion. It is thus tempting to speculate that the low expression and activation of NADPH oxidase in the hippocampal CA3/DG region after GCI reperfusion may contribute to its well known resistance to ischemic damage.
Our study also showed that E2 profoundly attenuates neuronal NADPH oxidase activation and O2− production in the CA1 region after GCI reperfusion. This effect appeared specific for the ischemic situation, because E2-treated sham animals did not show reduced NADPH oxidase activity or O2− levels compared with vehicle-treated sham controls. Additionally, the mechanism of action of E2 suppression of NADPH oxidase activation and O2− generation was shown to involve attenuation of Rac1 activation, which is known to be critical for NOX2 complex formation and activation. E2 did not significantly affect protein expression levels of NOX2, p47phox, p67phox, or Rac1 in the hippocampal CA1 region. Rather, E2 enhanced phosphorylation of Rac1 at Ser71, an effect that involved Akt mediation. Previous work has shown that phosphorylation of Rac1 at Ser71 leads to inhibition of Rac1–GTP binding, which is critical for Rac1 activation (Zhang et al., 2006b). In agreement with this, our study showed that E2 and EDC enhancement of p-Rac1 levels was correlated with reduced Rac1–GTP binding. NOX2 NADPH oxidase has also been demonstrated recently to have a significant role in Alzheimer's disease pathology (Block, 2008; Park et al., 2008), and thus our observed E2 mechanism of strongly inhibiting NADPH oxidase activation and ROS generation could also have relevance to E2 beneficial effects in Alzheimer's disease. This possibility awaits additional study. We should add that the effects of E2 on NADPH oxidase, Rac1, and Akt were shown to be ER mediated because they were blocked by acute treatment with the ER antagonist ICI182,780. The ability of acute ICI182,780 treatment to block E2 neuroprotection, in which E2 was administered for 1 week before GCI, suggests that E2 actions immediately after ischemia are critically important for the neuroprotective effect of E2. In particular, based on the results of our study, E2 action to modulate activation of prosurvival factors (e.g., Akt) and oxidative prodeath pathways (e.g., Rac1 and NADPH oxidase/ROS) early after cerebral ischemia appear key to the ability of E2 to exert neuroprotection in the hippocampal CA1 region, and ICI182,780 blocked these key E2 actions.
It should be noted that, although the CA3/DG region had high ERα levels, E2 had no significant effect on NADPH oxidase activation or O2− levels in this hippocampal region. However, the CA3/DG region did not display a significant reperfusion-induced increase in NADPH oxidase activation or O2− levels after stroke, which may explain the lack of significant E2 effect (e.g., there was no significant elevation for E2 to inhibit). Our study also provided evidence that extranuclear ERα-mediated signaling contributes significantly to E2 neuroprotective effects in the CA1 region after GCI. Use of EDC, a cell-impermeable E2 conjugate that interacts with extranuclear ER but not nuclear ER (Harrington et al., 2006), suppressed NADPH oxidase activation and O2− production but rapidly stimulated Akt activation and neuroprotection. EDC neuroprotection was blocked by the ER antagonist ICI182,780 and by the AkT inhibitor SH-5, demonstrating that its effects are ER mediated and involve Akt activation.
The antioxidant and neuroprotective effect of E2 in our study appeared to be mediated primarily by ERα, because antisense oligonucleotide knockdown of ERα but not ERβ resulted in a loss of NADPH oxidase and O2− regulatory effects and neuroprotection by E2. Furthermore, the antioxidant and neuroprotective effects of E2 were lost in long-term E2-deprived animals, which displayed a loss of ERα but not ERβ in the hippocampal CA1 region. Previous work had suggested that both ERα and ERβ may mediate E2 neuroprotective effects in the CA1 after GCI based on results using putative ER-selective agonists (Miller et al., 2005), but the selectivity of the agonists was not confirmed. Based on our studies using AS-ODN knockdown of each ER isoform, we conclude that ERα is the principal mediator of E2 neuroprotective actions in the hippocampal CA1 region after GCI.
Our study also provides important support for the critical period hypothesis for E2 beneficial effect (Maki, 2006; Sherwin, 2007a; Suzuki et al., 2007; Sherwin and Henry, 2008), because we observed that long-term E2 deprivation resulted in a complete loss of E2 nongenomic signaling, antioxidant actions, and neuroprotective effects in the hippocampal CA1 region. Intriguingly, the loss of E2 sensitivity was tissue specific, because the uterotrophic effect of E2 was not lost. Paralleling, and potentially explaining, the tissue-specific loss of E2 sensitivity was our observation of a loss of ERα (but not ERβ) in the hippocampus CA1 region but not in the uterus after long-term E2 deprivation. In previous work in the rat cerebral cortex, Suzuki et al. (2007) reported an ischemia-induced elevation of ERα in the cerebral cortex after MCAO, an effect that was lost in long-term E2-deprived rats. However, our results in the hippocampal CA1 region differ, because we observed a loss of ERα in the CA1 region in all groups (including sham controls), suggesting a loss of basal ERα independent of treatment or injury. A similar loss of basal ERα levels has been reported to occur in the aorta after prolonged E2 deprivation, suggesting that the loss of basal ERα occurs in both neural and vascular tissues (Pinna et al., 2008). Currently, it is unknown as to why ERα is lost in the hippocampal CA1 region but not in the uterus after prolonged hypoestrogenicity. Additional work is needed to address the mechanism of this tissue-specific loss of ERα.
Finally, an additional novel observation derived from our studies was that we observed a dramatically enhanced hypersensitivity of the hippocampal CA3/CA4 region to ischemic injury and neuronal cell death after long-term E2 deprivation. This is intriguing because long-term ovariectomy (surgical menopause) in humans has been correlated with an increased risk of cognitive decline and dementia, although the mechanisms underlying the effects have remained unclear (Rocca et al., 2007, 2008; Shuster et al., 2008). Our study may provide a mechanistic explanation for this increased risk by demonstrating a hypersensitivity of the hippocampal CA3/CA4 to injury after prolonged hypoestrogenicity. Sham animals showed no loss of CA3/CA4 neurons after prolonged hypoestrogenicity, suggesting that the ability of the CA3/CA4 to withstand a severe stress after prolonged hypoestrogenicity is severely compromised and that E2 deprivation itself, per se, does not cause loss of CA3/CA4 neurons. E2 treatment begun after prolonged hypoestrogenicity did not prevent the induction of the CA3/CA4 hypersensitivity, which may be attributable to a loss of E2 sensitivity of this region, as shown for the CA1 region. Additional studies are needed to determine whether E2 treatment initiated before the period of prolonged hypoestrogenicity would protect the CA3/CA4 region and prevent the induction of the ischemic hypersensitivity. One caveat of our studies is that young adult animals were used, and it is unclear whether aged animals would display a similar result. Thus, studies in aged animals are needed to further assess age-related alterations of E2 sensitivity and neuroprotection in the hippocampus. However, it should be mentioned that surgical menopause (ovariectomy) produces hypoestrogenicity at an early age in humans, which is similar to our model. Furthermore, surgical menopause in humans is associated with increased risk of cognitive decline, dementia, and cardiovascular disease (Shuster et al., 2008).
In conclusion, the current study advances the field by elucidating a novel and potent extranuclear ERα-mediated action of E2 in the brain to suppress NADPH oxidase activation and O2− generation in hippocampal CA1 neurons after stroke. It also provides important evidence supporting a critical period for E2 neuroprotective actions in the hippocampus and demonstrates a tissue-specific loss of E2 action and basal ERα expression, which may explain how and why the critical period exists. Finally, it demonstrates that the often ignored CA3/CA4 becomes hypersensitive to injury after long-term E2 deprivation, which may help explain the increased risk of cognitive decline and dementia observed after prolonged hypoestrogenicity or long-term ovariectomy in women.
This research was supported by National Institute of Neurological Diseases and Stroke/National Institutes of Health Research Grant NS050730.
- Correspondence should be addressed to Dr. Darrell W. Brann, Developmental Neurobiology Program, Institute of Molecular Medicine and Genetics, 1120 Fifteenth Street, Medical College of Georgia, Augusta, GA 30912.