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Abstract

17β-Estradiol (E₂) was shown to exert neuroprotective effects both in in vitro and in vivo models of stroke. Although these effects of E₂ are known to require estrogen receptor-α (ERα), the cellular target of estrogen-mediated neuroprotection remains unknown. Using cell type-specific ER mutant mice in an in vivo model of stroke, we specifically investigated the role of ERα in neuronal cells versus its role in the microglia in the mediation of neuroprotection by estrogens. We generated and analyzed two different tissue-specific knockout mouse lines lacking ERα either in cells of myeloid lineage, including microglia, or in the neurons of the forebrain. Both E₂-treated and E₂-untreated mutant and control mice were subjected to a permanent middle cerebral artery occlusion for 48 h, and the infarct volume was quantified. Although the infarct volume of E₂-treated female myeloid-specific ERα knockout mice was similar to that of E₂-treated control mice, both male and female neuron-specific ERα mutant mice had larger infarcts than did control mice after E₂ treatment. We conclude that neuronal ERα in female and male mice mediates neuroprotective estrogen effects in an in vivo mouse model of stroke, whereas microglial ERα is dispensable.

Keywords

cerebral ischemia estrogen receptor-α microglia neuron

Introduction

17β-Estradiol (E₂) has been shown to have protective effects against brain injury and neurodegeneration in several in vivo studies (Merchenthaler et al, 2003). 17β-Estradiol treatment of animals leads to smaller infarct size compared with untreated animals (Dubal et al, 1999). Studies in genetically modified animals showed that estrogen receptor-α (ERα) is essential for neuroprotection by estrogens in models of middle cerebral artery occlusion (MCAO) (Dubal et al, 2001). However, the cellular mechanism remains elusive, as several estrogen-responsive cell types could mediate this protective effect. In vitro studies showed direct protective actions of E₂ on neurons, e.g., in serum deprivation and glutamate-induced excitotoxicity (McEwen and Alves, 1999). Alternatively, microglial cells have been described to have an important role in inflammatory response and apoptosis in acute injury models of the brain both in vitro and in vivo (Lalancette-Hebert et al, 2007; Vegeto et al, 2003). Furthermore, microglial ERα exerts an important antiinflammatory effect in in vitro models of neuronal damage (Ghisletti et al, 2005). To dissect the neuroprotective action of estrogens on a cellular level, we generated two different cell type-specific ERα knockout mouse lines using the Cre-loxP system, deleting ERα in the microglia or in neurons of the brain, respectively, and subjected the mice to permanent focal cerebral ischemia.

Material and methods

Animals

All mouse strains were crossed to a C57BL/6 background. C57BL/6 mice were obtained from Charles River (Sulzfeld, Germany). Crossing the mice to a C57BL/6 background for four generations guarantees a nearly complete C57BL/6 background.

Myeloid-specific ERα mutant mice were generated by breeding mice expressing the Cre recombinase under control of the lysozyme M promoter (Clausen et al, 1999) with mice harboring loxP site-modified ERα alleles (ER^fl/fl) (Wintermantel et al, 2006). To generate mice lacking ERα in the neurons of the forebrain, mice expressing the Cre recombinase under control of the CaMKII promoter were bred with ER^fl/fl mice (Wintermantel et al, 2006).

Microglial Cell Culture

Microglial cells were isolated from newborn mice (P1) as described previously (Burudi et al, 1999). Flasks (one 75-cm² flask for three brain samples) were coated with poly-L-lysine (0.01%). The brains of 1-day-old mice were isolated and collected in a cell culture dish containing Hank's balanced salt solution. The meninges were removed. The brains were then homogenized and lysates were incubated for 20 mins at room temperature after adding 1 mL of 1% trypsin. After 20 mins, Falcon tubes containing the cell lysate were filled to a volume of 50 mL with complete medium (complete Dulbecco's modified Eagle's medium (cDMEM): DMEM, 10% fetal bovine serum, 100 Units/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L glutamine) and centrifuged for 10 mins at 180 g. The precipitate was resuspended with 1 mL cDMEM for every three brains. The cell lysate was distributed with 1 mL per flask and incubated at 37°C, 5% CO₂. The medium was changed at days 1, 2, and 7. After 14 days, the secondary culture for selection of microglial cells was performed. All steps were performed at room temperature. Flasks were shaken vigorously to detach oligodendrocytes and microglial cells bound to the surface of the cell layer. The medium was discarded, and the flasks were rinsed once with 10 mL cDMEM each and incubated with 3 mL trypsine-EDTA per flask for 3 mins at room temperature. Trypsine-EDTA was discarded and 10 mL of cDMEM and 0.5 mL of DNase were added. The cells were resuspended and centrifuged for 10 mins at 180 g. The cell precipitate was resuspended with 4 mL of cDMEM. A volume of 1 mL of the resuspended cells was added to a Petri dish and incubated at 37°C, 5% CO₂ for 20 mins. After 20 mins of incubation, 6 mL of cDMEM was added to the cultures and the cells were grown at 37°C, 5% CO₂. The medium was changed once a week. Microglial cells were used after 3 weeks in vitro for further analysis.

Estrogen Receptor-α Immunocytochemistry

Coverslips with attached microglial cells were washed with phosphate-buffered saline (PBS)/MgCl₂ and fixed with 4% paraformaldehyde. Thereafter, the cells were treated for 10 mins with 50 mmol/L NH₄Cl/PBS and permeabilized for 15 mins with 0.1% Triton-X-100/PBS. The cells were washed with PBS/MgCl₂ and incubated for 20 mins with 5% NSS (normal sheep serum). Incubation with anti-ERα antibody (polyclonal anti-ERα MC-20, Santa Cruz (Heidelberg, Germany) sc-542, diluted 1:500 in 5% NSS) was performed overnight at 4°C. The cells were then washed thrice for 5 mins and incubated for 30 mins with the secondary antibody (anti-rabbit AlexaFluor 594, Invitrogen (Karlsruhe, Germany), diluted 1:500 in 5% NSS). After washing the cells thrice for 5 mins with PBS/MgCl₂, they were incubated overnight with isolectin GS-IB₄ AlexaFluor 488 from Griffonia simplicifolia (Invitrogen, diluted 1:20 in 5% NSS) at 4°C. Finally, the cells were washed thrice for 5 mins with PBS/MgCl₂ and mounted with Mowiol (Roth, Karlsruhe, Germany). Estrogen receptor-α-positive cells and isolectin B₄-positive cells were counted using fluorescence microscopy. Recombination efficiency was determined by the ratio of ERα-positive to isolectin B₄-positive cells. Overall, 400 cells were evaluated. Microglial cells of ERα^fl/fl mice were stained as a control.

For detection of ERα in sections, the brains were fixed in 4% paraformaldehyde for 48 h. After blocking of endogenous peroxidase activity, coronal vibratome sections (40-μm-thick) were incubated overnight with anti-ERα. The primary antibody was detected with biotinylated anti-rabbit antibody, the ABC-peroxidase system (Vector, Burlingame, CA, USA), and 3,3′-diaminobenzidine (Sigma, Munich, Germany) as substrate.

Real-Time Reverse Transcription-PCR

Cortices were dissected and homogenized using an Ultra-Turrax T8 homogenizer (IKA Werke, Staufen, Germany). For RNA isolation, we used the RNeasy Mini Kit (Qiagen, Hilden, Germany). We removed genomic DNA from the lysate by using RNase-free DNase (Qiagen). RNA was transcribed using the SuperScript II Reverse Transcriptase Kit (Invitrogen). For real-time PCR, the Absolute QPCR-mix (Thermo Scientific, Karlsruhe, Germany) and the following primers and probes were used: ERα, Mm 00433149_m1; hypoxanthine guanine phosphoribosyl transferase (HPRT), Mm 00446968_m1 (Applied Biosystems, Darmstadt, Germany). Results were normalized to HPRT expression.

Middle Cerebral Artery Occlusion

Eight-week-old female mice were anesthetized with intraperitoneal injection of 150 μL of 2.5% tribromoethanol per 10 g body weight and ovariectomized. Both female and male mice received a pellet containing 0.025 mg E₂ with a release time of 21 days (Innovative Research of America, Sarasota, FL, USA). Control animals received a placebo pellet. Mice were randomly assigned to treatment groups. Ten days after ovariectomy and implantation of pellets, E₂ plasma levels were determined by a chemiluminescent immunoassay and the ADVIA centaur (Siemens, Nürnberg, Germany). 17β-Estradiol-treated ovariectomized mice had E₂ levels in the physiologic range (ovariectomized, E₂-treated, 68.4 ± 13.3 pg/mL, n = 7; female controls, 58.1 ± 4.7 pg/mL, n = 4). After 10 days of recovery from ovariectomy or implantation of the pellet, respectively, mice were anesthetized with intraperitoneal injection of 150 μL of 2.5% tribromoethanol per 10 g body weight. Middle cerebral artery occlusion was performed in mice as described previously (Herrmann et al, 2005; Zhang et al, 2005). Briefly, a skin incision was made between the ear and the orbit on the left side. The temporal muscle was removed by electrical coagulation. The stem of the MCA was exposed through a burr hole and was occluded by microbipolar coagulation. Mice were kept at a body temperature of 37°C on a heating pad. The body temperature was monitored continuously during the surgery using a rectal thermometer. To determine the infarct volume, mice were killed 48 h after the MCAO.

Coronal serial frozen sections of the forebrain were prepared. Silver staining was performed to determine the infarct volume as described previously (Schneider et al, 1999). Stained sections were scanned at 300 d.p.i., and the infarct area was measured using the Scion ImageJ software (Scion, Frederick, MD, USA). To correct for edema formation, the difference between areas of the left and right hemispheres was subtracted from the measured silver-negative area (Swanson et al, 1990). For calculating the whole brain infarct volume, the infarct areas were added and multiplied by the distance between the sections (0.4 mm). Surgery was performed, and ischemic damage was measured by an investigator who had no knowledge of the treatment group or the genotype. Animal experiments were approved by the Regierungspräsidium Karlsruhe.

Measurement of Physiologic Parameters

Measurement of arterial blood pressure, pulse, and blood gases was carried out both before and after ischemia in a separate cohort of mice. Mice were kept under tribromoethanol anesthesia on a heating pad at 37°C, and the body temperature was measured using a rectal thermometer. For measuring blood pressure and pulse, a cannula was inserted into the right femoral artery. A blood sample of 150 μL per mouse was collected in a heparin-coated glass capillary for analysis of arterial blood gas, hemoglobin, and glucose levels. The catheter was washed with 200 μL NaCl solution mixed with 50 IE (international units) of heparin before measuring blood pressure. For laser Doppler measurements, the electrode (P415-205; Perimed, Jarfalla, Sweden) was placed 3 mm lateral and 6 mm posterior to the bregma. Relative perfusion units were determined (Periflux 4001; Perimed).

Statistical Analysis

Student's t-test was used to compare two groups and one-way ANOVA (analysis of variance) for comparing more than two groups. Data are expressed as means ± s.d.

Results

17β-Estradiol treatment is neuroprotective in various rodent models of stroke (Gibson et al, 2006). To verify the efficacy of E₂ treatment in a model of permanent distal MCAO using C57BL/6 mice, we implanted ovariectomized female mice with E₂ or placebo pellets. Ten days later, mice were subjected to MCAO and killed after 48 h to determine the infarct volume. 17β-Estradiol treatment significantly reduced the infarct volume (Figure 1).

Figure 1

17β-Estradiol (E₂) reduced the infarct volume in ovariectomized mice that were subjected to permanent distal MCAO. The infarct volume was determined 48 h after MCAO. Means ± s.d. and individual values are depicted (n = 6 to 8). *P < 0.05 (t-test).

To investigate the role of ERα in myeloid cells including microglia, we generated myeloid lineage-specific ERα mutant mice by breeding mice expressing the Cre recombinase (Cre) under control of the lysozyme M promoter (Clausen et al, 1999) with mice harboring a conditional ERα allele (LysCre/ER^fl/fl) (Wintermantel et al, 2006). To investigate the recombination efficiency, we detected nuclear ERα expression in isolated microglial cells by immunocytochemistry. All the microglia obtained from control mice were stained (Figures 2A and 2C), whereas 92% of the microglial cells isolated from LysCre/ER^fl/fl mice showed no immunoreactivity for ERα (Figures 2B and 2C). 17β-Estradiol treatment reduced the infarct volume of control mice (ER^fl/fl). After E₂ treatments, the infarct size of LysCre/ER^fl/fl mice and control mice did not differ (Figure 3), indicating that the absence of microglial ERα does not affect ischemic brain damage in E₂-treated mice.

Figure 2

Deletion of ERα in microglial cells of LysCre/ER^fl/fl mice. (A–C) ERα is detected by immunohistochemistry (red signal) in isolated microglial cells obtained from ER^fl/fl mice (panel A), whereas no ERα expression is detectable in microglial cells isolated from LysCre/ER^fl/fl mice (panel B). Microglial cells were identified by isolectin B4 staining (green signal). (Panel C) All isolated microglial cells obtained from ER^fl/fl mice showed immunoreactivity for ERα, whereas only 8% of the isolated microglial cells obtained from LysCre/ER^fl/fl mice were positive for ERα. The color reproduction of this figure is available on the html full text version of the manuscript.

Figure 3

Deletion of ERα in microglial cells did not interfere with the neuroprotective effect of 17β-estradiol (E₂). The infarct volume of E₂-treated female LysCre/ER^fl/fl (n = 8) and control ER^fl/fl mice (n = 11) did not differ. In both groups, the infarct volume was significantly lesser than in female control ER^fl/fl mice treated with placebo pellets (n = 11). ANOVA, F(2/27) =5.739, P = 0.008. *P < 0.05 (Student–Newman–Keuls method) compared with ER^fl/fl-E₂. Means ± s.d. and individual values are depicted.

To analyze the contribution of neuronal ERα, we deleted ERα in the neurons of the forebrain by breeding mice expressing the Cre recombinase under control of the CaMKII promoter with ER^fl/fl mice (Wintermantel et al, 2006). As described previously for ERα expression in hypothalamic neurons, we found a marked reduction of ERα immunoreactivity in the cortices of CaMKIICre/ER^fl/fl mice (Figure 4A). Quantification of ERα mRNA by real-time reverse transcription PCR 24 h after MCAO confirmed this finding. Estrogen receptor-α mRNA was upregulated in the ischemic ipsilateral cortex compared with the contralateral side (Figure 4B) in accordance with a report that cerebral ischemia induces ERα expression (Dubal et al, 2006). However, in CaMKIICre/ER^fl/fl mice, ERα expression was markedly reduced both in the ischemic and the contralateral sides (Figure 4B).

Figure 4

Reduced ERα expression in CaMKIICre/ER^fl/fl mice. (A) ERα expression in the cortex was reduced in CaMKIICre/ERfl/fl mice as shown by immunohistochemistry. Upper row, ectorhinal cortex. Lower row, piriform cortex. (B) ERα mRNA levels in the ipsilateral ischemic and the contralateral cortex of ovariectomized ER^fl/fl and CaMKIICre/ER^fl/fl mice that were treated with 17β-estradiol (E₂) as indicated. mRNA was quantified by realtime RT-PCR 24 h after MCAO. ANOVA, F(5/52) = 20.297, P < 0.001. *P < 0.02 (Student–Newman–Keuls method). Means ± s.d. (n = 9 to 10) and individual values are depicted. The color reproduction of this figure is available on the html full text version of the manuscript.

17β-Estradiol-treated female CaMKIICre/ER^fl/fl mice showed an increased infarct volume compared with E₂-treated control mice 48 h after MCAO (Figures 5A and 5B). Infarct volumes of E₂-treated CaMKIICre/ER^fl/fl mice were similar to those of the placebo-treated control animals. This finding was reproduced by an independent experiment (infarct volume of ovariectomized ER^fl/fl mice without E₂ treatment, 21.0 ± 3.6 mm³, n = 7; ovariectomized ER^fl/fl mice with E₂ treatment, 16.0 ± 5.6 mm³, n = 8; ovariectomized CaMKII/ER^fl/fl with E₂ treatment, 24.5 ± 6.3 mm³ *, n = 7; ANOVA, F(2/19)=4.833, P = 0.020; *P < 0.05 compared with ER^fl/fl mice with E₂ treatment, Student–Newman–Keuls method). We conclude that the neuroprotective effect of E₂ is absent in female mice lacking ERα specifically and only in neurons.

Figure 5

Deletion of ERα in neurons abolished the neuroprotective effect of 17β-estradiol (E₂) in female mice. (A) Representative silver stainings of coronal brain sections of mice 48 h after MCAO. (B) E₂-treated female CaMKIICre/ER^fl/fl mice (n = 8) showed a clear increase in the infarct volume compared with E₂-treated female control ER^fl/fl mice (n = 9). The infarct volumes of the CaMKIICre/ER^fl/fl mice were similar to those of the placebo-treated female control mice (n = 9). ANOVA, F(2/23)=4.173, P < 0.05. *P < 0.05 (Student–Newman–Keuls method). Means ± s.d. and individual values are depicted.

To investigate whether this mechanism is sex-specific, E₂-treated male CaMKIICre/ER^fl/fl and control mice were also investigated in the MCAO model. Similar to female mice, E₂-treated male CaMKIICre/ER^fl/fl mice showed an increased infarct volume compared with E₂-treated control mice (Figure 6), suggesting that neuroprotection by E₂ depends on neuronal ERα.

Figure 6

Deletion of ERα in neurons abolished the neuroprotective effect of 17β-estradiol (E₂) in male mice. The infarct volumes of E₂-treated male CaMKIICre/ER^fl/fl mice (n = 11) were larger than those of E₂-treated male control ER^fl/fl (n = 8) mice. Placebo-treated control mice, n = 10. ANOVA, F(2/26) = 5.697, P < 0.01. *P < 0.01 (Student–Newman–Keuls method).

To exclude differences in cardiovascular parameters between control and CaMKII/ER^fl/fl mice responsible for the phenotype, we measured blood pressure, pulse, blood gases, and glucose levels before and after MCAO (Table 1). No significant differences were detectable between genotypes.

Table 1

Preischemic and postischemic physiologic parameters of female ERα^fl/fl and CaMKIICre/ER^fl/fl mice, both treated with E₂

Parameter	ERαf^l/fl		CaMKIICre/ER^fl/fl

	Before MCAO (n = 4)	After MCAO (n = 4)	Before MCAO (n = 3)	After MCAO (n = 3)
Mean arterial pressure (mm Hg)	58.5 ± 6.6	54.8 ± 7.4	57.3 ± 3.1	55.7± 2.1
Pulse (b.p.m.)	297 ± 6	306 ± 7	296 ± 7	304 ± 7
Body temperature (°C)	37.5 ± 0.0	37.5 ± 0.1	37.2 ± 0.2	37.4± 0.1
Cerebral blood flow (relative units)	211.5 ± 67.1	34.8 ± 16.0	287.3 ± 21.9	34.0± 12.5
Glucose (mg/dL)	262.5 ± 71.1	279.5 ± 70.5	228.7 ± 21.9	249.0 ±49.1
pO₂ (mm Hg)	79.9 ± 8.0	64.9 ± 13.9	82.1 ± 7.7	76.2 ± 10.5
pCO₂ (mm Hg)	53.4 ± 7.7	59.6 ± 8.5	49.2 ± 4.4	53.2 ±4.8
pH	7.23 ± 0.05	7.15 ± 0.06	7.25 ± 0.03	7.10 ± 0.00

b.p.m. beats per minute; ERα, estrogen receptor-α; MCAO, middle cerebral artery occlusion.

There were no significant differences in physiologic parameters between genotypes detectable (t-test).

Discussion

The neuroprotective effect of estrogens in physiologic doses is well established (Gibson et al, 2006; Strom et al, 2009), but the molecular and cellular mechanisms remain controversial. Although receptor-independent effects of E₂ have been reported (Culmsee et al, 1999), there is ample evidence that suggests that ERs mediate most of the neuroprotective effect. In line with this concept, the ER antagonist ICI182780 increased the infarct volume after MCAO (Sawada et al, 2000). Selective ER agonists have been used to define which of the two ERs reduces ischemic brain damage. These studies suggested that in models of global ischemia, ERβ is involved in neuroprotection (Carswell et al, 2004; Miller et al, 2005), but in focal cerebral ischemia only an ERα agonist was neuroprotective (Farr et al, 2007). However, a neuroprotective function of ERα has been challenged by the finding that after transient MCAO, the infarct size did not differ in control and ERα knockout mice (Sampei et al, 2000). In this study, mice were not ovariectomized. As ERα knockout mice have highly elevated serum levels of E₂ (Couse et al, 1995), the effect of ERα may have been compensated by an ERα-independent effect of high E₂ levels. Indeed, deficiency of ERα but not of ERβ abolished the protective effect of E₂ in ovariectomized mice subjected to focal cerebral ischemia (Dubal et al, 2001). Our data in CaMKIICre/ER^fl/fl mice support the neuroprotective role of ERα.

Estrogen receptor-α is expressed in many cells of the brain, including neurons, astrocytes, microglia, and endothelial cells. Therefore, the localization of ERα involved in the protection against ischemic damage is not self-evident. Numerous in vitro studies have approached the cellular basis of E₂ neuroprotection presenting evidence for the involvement of neurons (McEwen and Alves, 1999), astrocytes (Dhandapani and Brann, 2007), microglia (Bruce-Keller et al, 2000; Ghisletti et al, 2005), or endothelial cells (Razmara et al, 2008). In vivo E₂ inhibits microglia activation through ERα (Vegeto et al, 2003) and induces the expression of bfl-1, an antiapoptotic member of the bcl-2 gene family, in the microglia (Zhang et al, 2004). Whether these effects cause neuroprotection is unclear. To identify the cell type in which ERα exerts its neuroprotective action, we deleted ERα in neurons or microglia and performed MCAO in these mice.

The results show that ERα in the microglia and possibly in other cells of myeloid lineage does not mediate the effect of E₂ on infarct volume. We investigated the infarct volume 48 h after MCAO, when the microglia was activated in the MCAO model we used (Muhammad et al, 2008). However, we cannot exclude the fact that at other time points ERα deletion in myeloid cells may affect the infarct size.

In contrast to LysCre/ER^fl/fl mice, neuronal ERα knockout animals had larger infarcts than did controls after E₂ treatment. In fact, the infarct size of E₂-treated CaMKIICre/ER^fl/fl mice was identical to that of untreated controls. We cannot exclude that ERα exerts an E₂-independent effect on the infarct size. It is also conceivable that androgen levels were altered in male CaMKIICre/ER^fl/fl mice, as reported in ERα knockout mice (McDevitt et al, 2007), and that androgens may have affected ischemic brain damage, because male mice were not castrated in our experiments. However, the most likely explanation of our findings is that neuroprotection by E₂ is mediated by neuronal ERα. The introduction of the CaMKIICre gene to delete ERα from neurons is a possible confounding factor of our results. As previous experiments found no effect of other Cre genes on ischemic brain damage (Inta et al, 2006; data not shown), it seems very unlikely that the CaMKIICre gene itself could lead to enlarged infarct volumes.

This study suggests neuronal ERα as one of the major targets of E₂-mediated neuroprotection in a model of cerebral stroke. To our knowledge, this is the first time that the neuroprotective actions of ERα in stroke were investigated on a cellular level in vivo. It should be noted that cells of the vessel wall, glia, and hematopoietic cells retain ERα in the CaMKIICre/ER^fl/fl mouse line, but the E₂ action on these targets apparently does not influence the infarct size in this model of acute stroke. Clearly, potential beneficial vascular actions of ERα in stroke prevention and recovery are not addressed in this stroke model.

In view of these in vivo findings, previous studies analyzing the neuroprotective effects of estrogens in cultured neurons gain importance. Intracellular signaling pathways leading to neuroprotection involve PI3K, AKT, and MAP kinases (Morrison et al, 2006). In addition, ERs are known to inhibit the transcription factor nuclear factor-κB (Biswas et al, 2005), an effect that has also been reported in a model of cerebral ischemia in vivo (Wen et al, 2004). Inhibition of nuclear factor-κB in neurons is protective in stroke models (Herrmann et al, 2005; Zhang et al, 2005), suggesting that pathway-selective ER ligands that inhibit nuclear factor-κB (Chadwick et al, 2005) might be an option for stroke treatment. Another way to refine estrogen treatment for stroke therapy would be the development of neuron-specific selective ER modulators (neuro-SERMs) (Zhao et al, 2005), a strategy that would exploit the important role of neuronal ERα shown in this study.

Footnotes

TMW is an employee of Bayer Schering Pharma AG.

Acknowledgements

We are grateful to R Geibig and M Westphal for technical support.

References

Biswas

Singh

Shi

Pardee

Iglehart

(2005) Crossroads of estrogen receptor and NF-kappaB signaling. Sci STKE 2005:pe27

Bruce-Keller

Keeling

Keller

Huang

Camondola

Mattson

(2000) Antiinflammatory effects of estrogen on microglial activation. Endocrinology 141:3646–56

Burudi

Riese

Stahl

Regnier-Vigouroux

(1999) Identification and functional characterization of the mannose receptor in astrocytes. Glia 25:44–55

Carswell

HVO

Macrae

Gallagher

Harrop

Horsburgh

(2004) Neuroprotection by a selective estrogen receptor /{beta} agonist in a mouse model of global ischemia. Am J Physiol Heart Circ Physiol 287:H1501–4

Chadwick

Chippari

Matelan

Borges-Marcucci

Eckert

Keith

Jr Albert

Leathurby

Harris

Bhat

Ashwell

Trybulski

Winneker

Adelman

Steffan

Harnish

(2005) Identification of pathway-selective estrogen receptor ligands that inhibit NF-kappaB transcriptional activity. Proc Natl Acad Sci USA 102:2543–8

Clausen

Burkhardt

Reith

Renkawitz

Forster

(1999) Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgen Res 8:265–77

Couse

Curtis

Washburn

Lindzey

Golding

Lubahn

Smithies

Korach

(1995) Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene. Mol Endocrinol 9:1441–54

Culmsee

Vedder

Ravati

Junker

Otto

Ahlemeyer

Krieg

Krieglstein

(1999) Neuroprotection by estrogens in a mouse model of focal cerebral ischemia and in cultured neurons: evidence for a receptor-independent antioxidative mechanism. J Cereb Blood Flow Metab 19:1263–9

Dhandapani

Brann

(2007) Role of astrocytes in estrogen-mediated neuroprotection. Exp Gerontol 42:70–5

10.

Dubal

Shughrue

Wilson

Merchenthaler

Wise

(1999) Estradiol modulates bcl-2 in cerebral ischemia: a potential role for estrogen receptors. J Neurosci 19:6385–93

11.

Dubal

Zhu

Rau

Shughrue

Merchenthaler

Kindy

Wise

(2001) Estrogen receptor alpha, not beta, is a critical link in estradiol-mediated protection against brain injury. Proc Natl Acad Sci USA 98:1952–7

12.

Dubal

Rau

Shughrue

Zhu

Cashion

Suzuki

Gerhold

Bottner

Dubal

Merchanthaler

Kindy

Wise

(2006) Differential modulation of estrogen receptors (ERs) in ischemic brain injury: a role for ER/{alpha} in estradiol-mediated protection against delayed cell death. Endocrinology 147:3076–84

13.

Farr

Carswell

HVO

Gsell

Macrae

(2007) Estrogen receptor beta agonist diarylpropiolnitrile (DPN) does not mediate neuroprotection in a rat model of permanent focal ischemia. Brain Res 1185:275–82

14.

Ghisletti

Meda

Maggi

Vegeto

(2005) 17Beta-estradiol inhibits inflammatory gene expression by controlling NF-kappaB intracellular localization. Mol Cell Biol 25:2957–68

15.

Gibson

Gray

Murphy

Bath

(2006) Estrogens and experimental ischemic stroke: a systematic review. J Cereb Blood Flow Metab 26:1103–13

16.

Herrmann

Baumann

De Lorenzi

Muhammad

Zhang

Kleesiek

Malfertheiner

Köhrmann

Potrovita

Maegele

Beyer

Burke

Hasan

Bujard

Wirth

Pasparakis

Schwaninger

(2005) IKK mediates ischemia-induced neuronal cell death. Nat Med 11:1322–9

17.

Inta

Paxian

Maegele

Zhang

Pizzi

Spano

Sarnico

Muhammad

Herrmann

Inta

Baumann

Liou

H-C

Schmid

Schwaninger

(2006) Bim and Noxa are candidates to mediate the deleterious effect of the NF-{kappa}B subunit RelA in cerebral ischemia. J Neurosci 26:12896–903

18.

Lalancette-Hebert

Gowing

Simard

Weng

Kriz

(2007) Selective ablation of proliferating microglial cells exacerbates ischemic injury in the brain. J Neurosci 27:2596–605

19.

McDevitt

Glidewell-Kenney

Weiss

Chambon

Jameson

Levine

(2007) Estrogen response element-independent estrogen receptor (ER)-/{alpha} signaling does not rescue sexual behavior but restores normal testosterone secretion in male ER/{alpha} knockout mice. Endocrinology 148:5288–94

20.

McEwen

Alves

(1999) Estrogen actions in the central nervous system. Endocr Rev 20:279–307

21.

Merchenthaler

Dellovade

Shughrue

(2003) Neuroprotection by estrogen in animal models of global and focal ischemia. Ann NYAcad Sci 1007:89–100

22.

Miller

Jover

Cohen

Zukin

Etgen

(2005) Estrogen can act via estrogen receptor /{alpha} and /{beta} to protect hippocampal neurons against global ischemia-induced cell death. Endocrinology 146:3070–9

23.

Morrison

Brinton

Schmidt

Gore

(2006) Estrogen, menopause, and the aging brain: how basic neuroscience can inform hormone therapy in women. J Neurosci 26:10332–48

24.

Muhammad

Barakat

Stoyanov

Murikinati

Yang

Tracey

Bendszus

Rossetti

Nawroth

Bierhaus

Schwaninger

(2008) The HMGB1 receptor RAGE mediates ischemic brain damage. J Neurosci 28:12023–31

25.

Razmara

Sunday

Stirone

Wang

Krause

Duckles

Procaccio

(2008) Mitochondrial effects of estrogen are mediated by estrogen receptor /{alpha} in brain endothelial cells. J Pharmacol Exp Ther 325:782–90

26.

Sampei

Goto

Alkayed

Crain

Korach

Traystman

Demas

Nelson

Hurn

(2000) Stroke in estrogen receptor-alpha-deficient mice. Stroke 31:738–43; discussion 44

27.

Sawada

Alkayed

Goto

Crain

Traystman

Shaivitz

Nelson

Hurn

(2000) Estrogen receptor antagonist ICI182780 exacerbates ischemic injury in female mouse. J Cereb Blood Flow Metab 20:112–8

28.

Schneider

Martin-Villalba

Weih

Vogel

Wirth

Schwaninger

(1999) NF-κB is activated and promotes cell death in focal cerebral ischemia. Nat Med 5:554–9

29.

Strom

Theodorsson

(2009) Dose-related neuroprotective versus neurodamaging effects of estrogens in rat cerebral ischemia: a systematic analysis. J Cereb Blood Flow Metab 29:1359–72

30.

Swanson

Morton

Tsao-Wu

Savalos

Davidson

Sharp

(1990) A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab 10:290–3

31.

Vegeto

Belcredito

Etteri

Ghisletti

Brusadelli

Meda

Krust

Dupont

Ciana

Chambon

Maggi

(2003) Estrogen receptor-alpha mediates the brain antiinflammatory activity of estradiol. Proc Natl Acad Sci USA 100:9614–9

32.

Wen

Yang

Liu

Perez

Koulen

Simpkins

(2004) Estrogen attenuates nuclear factor-kappa B activation induced by transient cerebral ischemia. Brain Res 1008:147–54

33.

Wintermantel

Campbell

Porteous

Bock

Grone

Todman

Korach

Greiner

Perez

Schutz

Herbison

(2006) Definition of estrogen receptor pathway critical for estrogen positive feedback to gonadotropin-releasing hormone neurons and fertility. Neuron 52:271–80

34.

Zhang

Nair

Krady

Corpe

Bonneau

Simpson

Vannucci

(2004) Estrogen stimulates microglia and brain recovery from hypoxia-ischemia in normoglycemic but not diabetic female mice. J Clin Invest 113:85–95

35.

Zhang

Potrovita

Tarabin

Herrmann

Beer

Weih

Schneider

Schwaninger

(2005) Neuronal activation of NF-κB contributes to cell death in cerebral ischemia. J Cereb Blood Flow Metab 25:30–40

36.

Zhao

O'Neill

Diaz Brinton

(2005) Selective estrogen receptor modulators (SERMs) for the brain: current status and remaining challenges for developing Neuro-SERMs. Brain Res Rev 49:472–93

Neuronal Estrogen Receptor-α Mediates Neuroprotection by 17β-Estradiol

Abstract

Keywords

Introduction

Material and methods

Animals

Microglial Cell Culture

Estrogen Receptor-α Immunocytochemistry

Real-Time Reverse Transcription-PCR

Middle Cerebral Artery Occlusion

Measurement of Physiologic Parameters

Statistical Analysis

Results

Discussion

Footnotes

Acknowledgements

References