estradiol enhances neurogenesis following ischemic stroke through estrogen receptors α and β

Estradiol Enhances NeurogenesisFollowing Ischemic Stroke Through

Estrogen Receptors � and �

SHOTARO SUZUKI,1,2 LYNNETTE M. GERHOLD,1 MARTINA BOTTNER,1

SHANE W. RAU,3 CHRISTOPHER DELA CRUZ,1 ENHUA YANG,1 HONG ZHU,4

JIN YU,4 ADRIENNE B. CASHION,1 MARK S. KINDY,4 ISTVAN MERCHENTHALER,5

FRED H. GAGE,6AND PHYLLIS M. WISE1,2*

1Department of Neurobiology, Physiology and Behavior, University of California Davis,Davis, California 95616

2Department of Physiology and Biophysics, University of Washington,Seattle, Washington 98195

3Department of Physiology, College of Medicine, University of Kentucky, Lexington,Kentucky 40536

4Department of Physiology and Neuroscience, Medical University of South Carolina,Charleston, South Carolina 29425

5Department of Epidemiology and Preventive Medicine, University of Maryland,Baltimore, Baltimore, Maryland 21201

6Salk Institute for Biological Sciences, Laboratory of Genetics, La Jolla, California 92037

ABSTRACTNeurogenesis persists throughout life under normal and degenerative conditions. The adult

subventricular zone (SVZ) generates neural stem cells capable of differentiating to neuroblastsand migrating to the site of injury in response to brain insults. In the present study, weinvestigated whether estradiol increases neurogenesis in the SVZ in an animal model of stroke topotentially promote the ability of the brain to undergo repair. Ovariectomized C57BL/6J micewere implanted with capsules containing either vehicle or 17�-estradiol, and 1 week later theyunderwent experimental ischemia. We utilized double-label immunocytochemistry to identify thephenotype of newborn cells (5-bromo-2�-deoxyuridine-labeled) with various cellular markers;doublecortin and PSA-NCAM as the early neuronal marker, NeuN to identify mature neurons,and glial fibrillary acidic protein to identify astrocytes. We report that low physiological levels ofestradiol treatment, which exert no effect in the uninjured state, significantly increase thenumber of newborn neurons in the SVZ following stroke injury. This effect of estradiol is limitedto the dorsal region of the SVZ and is absent from the ventral SVZ. The proliferative actions ofestradiol are confined to neuronal precursors and do not influence gliosis. Furthermore, we showthat both estrogen receptors � and � play pivotal functional roles, insofar as knocking out eitherof these receptors blocks the ability of estradiol to increase neurogenesis. These findings clearlydemonstrate that estradiol stimulates neurogenesis in the adult SVZ, thus potentially facilitatingthe brain to remodel and repair after injury. J. Comp. Neurol. 500:1064–1075, 2007.© 2006 Wiley-Liss, Inc.

Indexing terms: BrdU; estrogen receptor, immunocytochemistry; ischemia; neural stem cells;

stroke; subventricular zone

Grant sponsor: National Institutes of Health; Grant number: AG17164(to P.M.W.); Grant sponsor: Ellison Foundation (to P.M.W.); Grant sponsor:Maryland Veteran Administration REAP Pilot Award (to I.M.).

*Correspondence to: Phyllis M. Wise, PhD, Provost and Vice Presidentfor Academic Affairs, University of Washington, 301 Gerberding Hall, Box351237, Seattle, WA 98195-1237. E-mail: [email protected]

Received 10 March 2006; Revised 18 July 2006; Accepted 19 September2006

DOI 10.1002/cne.21240Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 500:1064–1075 (2007)

© 2006 WILEY-LISS, INC.

One of the most exciting discoveries in modern neuro-science is that the mammalian brain is plastic and con-tinues to generate new neurons throughout adulthood un-der normal as well as degenerative conditions (Zhang etal., 2001; Jin et al., 2001; Arvidsson et al., 2002). It iscritical to identify the mechanisms that promote adultneurogenesis in the face of brain insults, because suchknowledge may allow the development of therapeuticstrategies for neuronal replacement in neurodegenerativediseases and injury. Several factors influence the capacityof the adult brain to generate neurons, including growthfactors (Yoshimura et al, 2001; Gustafsson et al., 2003),endogenous neuronal activity (Arvidsson et al., 2001), andenvironmental enrichment (Komitova et al., 2002). Hor-monal milieu also appears to influence neurogenesis: theneurogenic response varies with the reproductive stagesin the subventricular zone (SVZ) of prairie voles (Smith etal., 2001), and ovarian hormones enhance neurogenesis inthe hippocampus of rats (Tanapat et al., 1999).

The forebrain SVZ lining the lateral ventricle is a majorrepository of neural stem cells in the adult brain (Gross,2000; Gage, 2002; Alvarez-Buylla and Garcia-Verdugo,2002; Taupin and Gage, 2002). Most cells born in theanterior portion of the SVZ give rise to neuroblasts thatnormally migrate along the rostral migratory stream(RMS) to the olfactory bulb, where they differentiate intofunctional olfactory interneurons (Lois and Alvarez-Buylla, 1994). However, pathological processes induced bycortical aspiration, traumatic brain injury, and cerebralischemia redirect the migration of neuroblasts from theirnormal route toward the sites of injury, including thecortex and striatum (Arvidsson et al., 2002; Jin et al.,2003; Goings et al., 2004; Zhang et al., 2004; Ramaswamyet al., 2005; Sundholm-Peters et al, 2005; Tonchev et al.,2005). At the sites of pathology, the SVZ-derived neuro-blasts differentiate into mature neurons and form syn-apses with neighboring cells (Yamashita et al., 2006).Thus, identifying endogenous factors capable of stimulat-ing neurogenesis in the face of brain insults as well asunderstanding the mechanisms of their actions may pro-mote cell replacement therapies for neurodegenerativeconditions that often accompany neuronal loss.

During the past decade, we have begun to appreciatethat estradiol plays an important trophic as well as pro-tective role in the adult brain. Estradiol is essential notonly to maintaining normal brain function but also toprotecting the brain against neurodegenerative diseasesand injury (for reviews see Yaffe et al., 1998; McEwen andAlves, 1999; Behl, 2002). However, recent findings fromthe Women’ Health Initiative call into question whetherestrogens might under some circumstances actually in-crease the risk of neurodegeneration (Rapp et al., 2003;Shumaker et al., 2003, 2004; Wasserteil-Smoller et al.,2003). Therefore, it becomes even more critical to investi-gate when these hormones are beneficial and their mech-anisms of action. Both in vitro and in vivo studies thatutilize a variety of animal models show that estradiolprevents cell death, promotes neuronal survival, enhancesneurite outgrowth, stimulates synaptogenesis, and regu-lates synthesis of neurotransmitters and their receptorsunder a wide range of experimental paradigms (Wise etal., 2001). Thus, previously, we have shown that 17�-estradiol exerts profound neuroprotective actions in amodel of stroke injury in which the middle cerebral arteryis permanently occluded (Dubal et al., 1998, 2001). Estra-

diol achieves these actions by altering the expression ofmultiple genes involved in cell death/survival pathways(Rau et al., 2003a) and inhibiting the activity of caspases(Rau et al., 2003b), leading to increased neuronal survivalin the penumbra that surrounds the ischemic core.

The purpose of the present study was to investigatewhether an additional mechanism by which estradiol lim-its the extent of brain injury and further enhances brainrepair is by increasing the number of newborn neurons inthe adult SVZ after ischemic injury. The SVZ is the birth-place of neurons capable of migrating to the cortex andstriatum (Arvidsson et al., 2002; Jin et al., 2003; Goings etal., 2004; Zhang et al., 2004; Ramaswamy et al., 2005;Sundholm-Peters et al, 2005; Tonchev et al., 2005), twobrain regions that are most affected by stroke injury in-duced by middle cerebral artery occlusion (MCAO). There-fore, we examined the level of neurogenesis in the SVZafter permanent MCAO in ovariectomized C57BL/6J micepretreated with either vehicle or 17�-estradiol for 1 week.Our data reveal that low physiological levels of estradiolincrease the number of newborn neurons in the SVZ afterstroke-like injury and that both estrogen receptor (ER)-�and ER� are essential mediators. In the present study, weuncovered a dose of estradiol that does not stimulate pro-liferation of astrocytes or microglia and stimulates neuro-genesis only after injury and not in the absence of injury.

MATERIALS AND METHODS

All surgical procedures were performed in strict compli-ance with the National Institutes of Health Guide for thecare and use of laboratory animals and were approved bythe Institutional Animal Care and Use Committee at theUniversity of Kentucky, Chandler Medical Center, andthe University of California, Davis.

Estradiol replacement

In total, 90 adult female C57BL/6J mice (age 11 weeks,weight 19–22 g) were purchased from The Jackson Labo-ratory (Bar Harbor, ME), and an additional 30 age-matched ER� null (ER�–/–) and ER� null (ER�–/–) micewere obtained from WHRI, Wyeth Research (Collegeville,PA). These two strains of knockout mice had been back-crossed for at least eight generations on a C57BL/6J ge-netic background, and their generation has been describedpreviously (Couse et al., 1995; Dubal et al., 2001). Micewere ovariectomized to eliminate endogenous ovarian ste-roid production and implanted subcutaneously with a 20-mm-long Silastic capsule (0.062 in./0.125 in., inner/outerdiameter; volume, 0.035 ml; Konigsberg Instruments,Pasadena, CA) containing either sesame oil (vehicle) or17�-estradiol (180 �g/ml). This paradigm of estradioltreatment produces stable levels of 17�-estradiol in serum(25 pg/ml) equivalent to low-basal circulating levels foundduring the estrous cycle of mice (Dubal et al., 2001).

In vivo cerebral ischemia

One week after ovariectomy, animals were anesthetizedwith a mixture of chloral hydrate (350.0 mg per kg bodyweight, i.p.) and xylazine (4.0 mg/kg, i.p.), and the rightmiddle cerebral artery was permanently occluded as pre-viously describe (Dubal et al., 2001). Briefly, a 5/0-sizeblue nylon suture was inserted into the internal carotidartery to the base of the middle cerebral artery. Thisocclusion leads to a dramatic reduction in blood flow to the

The Journal of Comparative Neurology. DOI 10.1002/cne

1065E2 ENHANCES NEUROGENESIS AFTER STROKE INJURY

striatum and overlying cortex. Sham-operated mice un-derwent the same surgical procedure except that the ar-tery was not occluded. In all mice, body temperature wasmonitored and maintained at normothermia until recov-ery from anesthesia.

5-Bromo-2�-deoxyuridine injections

Adult mice were injected intraperitoneally with5-bromo-2�-deoxyuridine (BrdU; 50 mg/kg body weight) 1hour prior to MCAO and subsequently twice daily untilthey were killed either 24 or 96 hours later.

Specificity of antibodies

The specificity of monoclonal rat anti-BrdU (AccurateChemical, Westbury, NY; catalog No. OBT0030; lot No.H5903) was tested by performing immunocytochemistryon mice that did not receive BrdU injections. No specificstaining was obtained in brain sections from negativecontrol animals (no BrdU injections; data not shown). Anaffinity-purified polyclonal goat anti-Dcx (Santa Cruz Bio-technology, Santa Cruz, CA; catalog No. SC-8066; lot No.K011) raised against a peptide sequence corresponding toamino acids 385–402 of human Dcx was chosen on thebasis of its established specificity (Farrar et al., 2005;manufacturer’s technical information). This antiserum de-tects a single band of 40 kDa consistent with the molecu-lar weight of the Dcx protein on Western blot (Brown etal., 2003) and is specific for mouse, rat, and human Dcxrevealed by Western blot, immunoprecipitation, and im-munohistochemistry (Farrar et al., 2005; manufacturer’stechnical information). Monoclonal mouse anti-PSA-NCAM (Chemicon, Temecula, CA; catalog No. MAB5324;lot No. 22060929) also detects a singe band on Westernblot according to manufacturer’s technical information.Purified monoclonal mouse anti-NeuN (Chemicon; catalogNo. MAB337; lot No. 21070547) was raised against puri-fied cell nuclei from mouse brain (Yang et al., 2005). Poly-clonal rabbit antiglial fibrillary acidic protein (GFAP;Sigma, St. Louis, MO; catalog No. G9269; lot No.042K4895) was developed against GFAP purified fromhuman brain. This antiserum detects a single band of 46kDa on Western blot (manufacturer’s technical informa-tion). Polyclonal rabbit anti-ER� (Upstate Biotechnolo-gies, Lake Placid, NY; catalog No. 06-935; lot No. 21382)was raised against the last 15 amino acids of rat ER�(TYYIPPEAEGFPNTI). This antiserum detects a singleband of an expected molecular weight of 66 kDa on West-ern blot according to manufacturer’s technical informa-tion. The specificity of polyclonal rabbit anti-ER� (ZymedLaboratories, San Francisco, CA; catalog No. 51-7900; lotNo. 20470590) has been previously tested by several con-trol experiments including a preabsorption study, as wellas a dual-label immunocytochemistry/in situ hybridiza-tion technique to evaluate the colocalization of ER� im-munoreactivity and ER� mRNA (Shughrue and Merch-enthaler, 2001). This antiserum was raised against theC-terminus of the mouse ER� protein (CSTEDSKSKEG-SQNLQSQ). It detects a single band of 60 kDa on Westernblot (Shughrue and Merchenthaler, 2001).

Fluorescent immunocytochemistry

Animals were anesthetized with a mixture of chloralhydrate (350.0 mg/kg) and xylazine (4.0 mg/kg) and per-fused with saline, followed by 4% phosphate-bufferedparaformaldehyde, pH 7.0, containing 2% acrolein at 24 or

96 hours after MCAO. The brains were postfixed over-night at 4°C then stored in 30% sucrose until permeated.A series of free-floating 35-�m coronal sections was neu-tralized with 1% sodium borohydrate, denatured in 50%formamide/2� SSC for 2 hours at 65°C, treated with 2 NHCl, and incubated in blocking buffer (6% normal donkeyserum in PBS). The tissue was then incubated with ratanti-BrdU (Accurate Chemical; 1:400) for 72 hours at 4°C.Subsequently, sections were incubated with Alexa Fluor488 donkey anti-rat immunoglobulins (Molecular Probes,Eugene, OR; 1:1,500) for 2 hours at 25°C. Double-labelimmunocytochemistry was essentially similar to that de-scribed above. After blocking, sections were initially incu-bated with one of the following antisera; goat antidou-blecortin (Dcx; Santa Cruz Biotechnology; 1:300), mouseantipolysialylated neural adhesion molecule (PSA-NCAM;Chemicon; 1:400), mouse anti-NeuN (Chemicon; 1:100),rabbit anti-GFAP (Sigma; 1:10,000), or biotinylated to-mato lectin (Sigma; catalog No. L-2140; lot No. 62K4039;1:250). After incubation in primary antisera, correspond-ing secondary antibodies were used; Alexa Fluor 568 don-key anti-goat immunoglobulins (1:1,500), donkey anti-mouse Cy3 immunoglobulins (Jackson Immunoresearch,West Grove, PA; 1:500), donkey anti-rabbit Cy3 immuno-globulins (Jackson Immunoresearch; 1:500), streptavidin/Cy3 (Jackson Immunoresearch; 1:500), or streptavidin/cy3(for biotinylated tomato lectin; Jackson Immunoresearch;1:500). Next, sections were washed and subsequently pro-cessed for BrdU staining as described above.

We used Dcx, a microtubule-associated protein associ-ated with migrating neurons, as the principal marker ofan immature neuronal phenotype because this markerappears to be important for the normal developmentalmigration of cortical neurons. Dcx is also expressed insome apparently mature neurons in the adult brain,where it may be involved in axonal outgrowth or synap-togenesis. Additional cellular markers were used to iden-tify the phenotype of newborn cells: PSA-NCAM as theearly neuronal marker, NeuN to identify mature neurons,GFAP to identify astrocytes, and tomato lectin to identifymicroglia.

TUNEL staining

TUNEL assay was performed on brain sections accord-ing to the instructions of the manufacturer (in situ celldeath detection kit, fluorescein; Roche Products, Hertfor-shire, United Kingdom), as previously described (Rau etal., 2003b). Coronal mouse brain sections were fixed for 5minutes in 4% paraformaldehyde and subsequently incu-bated in permeabilization solutions (0.2% Triton X-100and 0.1% sodium citrate) for 2 hours at 37°C. Sectionswere then washed twice in phosphate-buffered saline(PBS), pH 7.0, followed by incubation for 60 minutes at37°C in TUNEL reaction mix from the Roche Products kit.Sections were then rinsed in distilled water and cover-slipped with antifade mounting medium.

Confocal microscopy and cell counting

Confocal images were obtained on a Leica DMIRB/Elaser scanning microscope (Leica Microsystems, Wetzlar,Germany), as previously described (Gerhold et al., 2002).All images were acquired in dual-scan mode with a z-scaninterval of 1 �m (�35 scans per stack). The confocal stackswere converted to eight-bit images, and stacks for eachcolor were combined, resulting in one RGB stack. For all


1066 S. SUZUKI ET AL.

animals, anatomical landmarks were identified accordingto the atlas of Paxinos and Franklin (2001) and used toselect the most representative section (one section permouse). The section was chosen at the level of bregma0.7–1.2 mm, because our previous study demonstratedthat estradiol exhibits most apparent protective actionsagainst ischemic injury at this level of the mouse brain

(data not shown). Subsequently, individual single-labeledcells were examined in each scan of the RGB color-combined stack and counted within the dorsal SVZ (be-tween bregma 0.7–1.2 mm; indicated by a pair of blackvertical lines in Fig. 1A; counted from a boxed area shownin Fig. 1B) in Metamorph image analysis software (ver-sion 6.0; Universal Imaging Corporation, Downingtown,

Fig. 1. Estradiol enhanced neurogenesis in the SVZ at 96 hoursafter the onset of MCAO. Schematic diagrams depicting the adultmouse brain in sagital (A) and coronal (B) views; cells were counted incoronal sections (between bregma 0.7 and 1.2 mm; denoted by a pairof black vertical lines in A) in the anterior portion of the dorsal(indicated in areas shown in orange) or the ventral (shown in a redsquare in B) SVZ. The representative infarct areas from oil (green)-and estradiol (blue)-treated mice that underwent permanent MCAOof the right hemisphere (Dubal et al., 2001) are indicated by green and

blue dashed lines, respectively. Estradiol increased the number ofBrdU-labeled and BrdU/Dcx dual-labeled cells in the SVZ at 96 hoursafter MCAO injury. Confocal photomicrographs of BrdU-labeled(green) and Dcx-labeled (red) cells in the ipsilateral dorsal SVZ (indi-cated by a dashed square in B) in oil-treated (C–E) vs. estradiol-treated (F–H) animals. Estradiol pretreatment increased the num-bers of BrdU-labeled as well as BrdU/Dcx dual-labeled cells. CC,corpus callosum; OB, olfactory bulb; RMS, rostral migratory stream;STR, striatum. Scale bar � 100 �m.



PA) without the knowledge of treatment groups. BrdU/Dcx dual-labeled cells were similarly analyzed within thesame region (dorsal SVZ between bregma 0.7 and 1.2 mm)after identifying an immunopositive cell (Dcx-positive)that contained an immunopositive nucleus (BrdU-positive) in Metamorph. Colocalization was verified byanalyzing each scan of the RGB color-combined stack forindividual cells (Fig. 2). The numbers were then correctedby using the equation of Abercrombie (1946), i.e., N � n(T/T � D), where N is the corrected cell numbers, n is theestimated raw cell counts, T is the section thickness, andD is the diameter of the object counted. The double-labeled

cells were identified as cells containing a green nucleus(BrdU-positive) and a red cytoplasm and nerve fibers(Dcx-positive). All images were formatted in Adobe Pho-toshop 7.0 (Adobe, San Jose, CA). In some figures, bright-ness and contrast settings were slightly adjusted.

Immunodetection of ER� and ER�

The primary antibody titer, the primary antibody incu-bation time and temperature, and developing time wereinitially varied, and optimal conditions were used insubsequent studies. In addition, we used avidin-biotin-peroxidase methods and nickel-intensified 3,3�-

Fig. 2. Confocal photomicrographs of BrdU-labeled cells (green)doubly stained with the early neuronal marker Dcx (red) in theipsilateral dorsal SVZ of estradiol-treated animals at 96 hours afterMCAO injury. Images shown in A–I were acquired in dual-scan mode

with a z-scan interval of 1 �m (each stack represents a 1-�m section)to illustrate the extent of colocalization. An image shown in J repre-sents confocal stacks of nine images (A–I). Arrows indicate represen-tative double-labeled cells. Scale bar � 50 �m.



diaminobenzidine as chromogen to optimize visualiza-tion of low-expression antigens. A series of free-floatingcoronal sections was neutralized with 1% sodium boro-hydrate; washed three times for 15 minutes each in 0.1M PBS with 0.1% Triton-X (TX), pH 7.0; and then incu-bated with 0.3% H2O2 in 0.1 M PBS with 0.1% TX for 15minutes to quench any endogenous peroxidase activity.Sections were subsequently incubated in blocking buffer(6% normal goat serum in PBS) to block any nonspecificantibody binding and then incubated for 72 hours at 4°Cwith ER� (Upstate Biotechnologies; 1:10,000) or ER�antiserum (Zymed Laboratories; 1:3,000) in 0.1 M PBSwith TX in the presence of 2% normal goat serum, aspreviously described (Suzuki and Handa, 2005). Next,the tissue was washed three times for 10 minutes eachin PBS with TX and incubated with biotinylated goatanti-rabbit IgG (Vector Laboratories, Burlingame, CA;1:500) in PBS with TX in the presence of 2% normal goatserum for 2 hours at room temperature. Sections weresubsequently washed and processed according to theavidin-biotin-peroxidase procedure (Vector Laborato-ries; 1:500). After standard washes, the tissue wasrinsed in 0.1 M Tris-buffered saline, pH 8.0, for 15minutes and then developed with nickel-intensified3,3�-diaminobenzidine (0.5 mg/ml; Sigma) in 0.1 M Tris-buffered saline containing 0.03% hydrogen peroxide.The reaction was stopped by several washes in 0.1 MPBS. The sections were subsequently mounted on Su-perfrost Plus glass slides (Fisher Scientific, Pittsburgh,PA), air dried for 24 hours at room temperature, furtherdehydrated through a series of increasing alcohols,cleared with xylene, and coverslipped with Permount(Fisher Scientific).

Data analysis

All data are expressed as mean SEM. The numbers ofBrdU-labeled and BrdU/Dcx dual-labeled cells were ana-lyzed via two-way analysis of variance (ANOVA), followedby the Bonferroni test. The effect of genotypes on cellcounts in estradiol-treated mice and the effect of injury onBrdU-labeled cell counts in oil-treated animals were ana-lyzed via one-way ANOVA, and significance was probed bythe Newman-Keuls test. All differences were consideredsignificant at P 0.05.

RESULTS

Estradiol increases the number of newborncells in the SVZ at 96 hours after MCAO

MCAO-induced injury significantly reduced the numberof BrdU-labeled cells in vehicle-treated mice at 96 hoursafter the onset of injury on both the ipsilateral and thecontralateral sides of the SVZ (#P 0.05, n � 6–7; Fig.3A). Pretreatment with estradiol significantly increasedthe number of BrdU-labeled cells after MCAO comparedwith vehicle-treated control mice on both the ipsilateral(Fig. 1C,F) and the contralateral (data not shown) sides ofthe SVZ (*P 0.001, n � 6–7; Fig. 3A). The estradiol-induced increase in the number of proliferating cells wasslightly more pronounced on the injured side of the brainthan on the contralateral side (the number of BrdU-labeled cells; ipsilateral oil, 86.3 8.10, E2, 174.3 33.17*; contralateral oil, 94.0 8.34, E2, 152.8 22.98*).It is important to point out that this effect of estradiol

occurs bilaterally, despite the fact that neuronal cell deathoccurs only on the side of artery occlusion (Dubal et al.,1998, 2001). We found that estradiol increased the num-ber of BrdU-labeled cells only in the dorsal portion of theSVZ and not in the ventral SVZ (indicated by a red squarein Fig. 1B; the number of proliferative cells in the ventralportion of the SVZ; ipsilateral oil, 54.8 15.06, E2, 56.1 10.54; contralateral oil, 38.4 7.69, E2, 46.6 8.29,mean SEM of 6–7 mice per group).

In marked contrast to the case in injured mice, the useof our paradigm of hormone administration showed thatthese low physiological levels of estradiol did not affectbasal proliferation in sham-operated mice (Fig. 3A). Thus,this dose of estradiol modulates the numbers of BrdU-labeled cells only in the face of injury and not in theuninjured SVZ.

Fig. 3. Estradiol enhanced proliferation as well as neurogenesis inthe SVZ at 96 hours after MCAO-induced injury. Estradiol increasedthe number of BrdU-labeled proliferating cells at 96 hours afterMCAO compared with oil-treated controls on the injured and unin-jured sides of the dorsal SVZ (*P � 0.0008, n � 6–7), but not in thesham-operated animals (A). In addition, ischemic injury bilaterallyreduced the number of BrdU-labeled cells at 96 hours after the onsetof injury in the dorsal SVZ in oil-treated mice (#P 0.05, n � 6–7).Estradiol significantly (*P 0.004, n � 6–7) increased the number ofnewborn neurons (BrdU/Dcx dual-labeled cells) in the dorsal SVZ inMCAO-injured mice at 96 hours (B). The numbers represent the meanof cells counted in one section of the brain per animal. In both series,data represent the mean SEM of six or seven animals per group.



Newborn cells in the dorsal SVZ express themarkers of immature neurons

To identify the phenotype of newly generated cells in thedorsal SVZ, we performed double-label fluorescent immu-nocytochemistry with neuronal and glial markers:; Dcx orPSA-NCAM as the early neuronal markers, NeuN to iden-tify mature neurons, GFAP to identify astrocytes, andlectin to identify microglia. Confocal laser scanning micro-scopic analysis revealed that the majority of BrdU-labeledcells coexpressed the markers of immature neurons, Dcx(ipsilateral oil, 67.93% 5.625%, E2, 68.39% 4.776%,Fig. 1C–H, Fig. 3; contralateral oil, 73.60% 1.974%, E2,74.95% 1.848%, data not shown) and PSA-NCAM (seeFig. 4A–C), but not the markers of mature neurons, suchas NeuN (see Fig. 4D–F). In addition, BrdU-labeled cells

in the SVZ did not coexpress the astrocyte marker GFAP(see Fig. 4G–I) or the microglial marker lectin (data notshown). As with overall proliferation, estradiol signifi-cantly increased the number of BrdU/Dcx dual-labeledcells (*P 0.004, n � 6–7) in the SVZ (Fig. 3B). Most ofthese cells displayed elongated processes, a feature ofmigrating neurons. Taken together, these findings sug-gest that the majority of newborn cells will differentiateinto neurons and that estradiol specifically targets thiscell type.

Estradiol does not influence apoptotic celldeath in the SVZ after MCAO-induced injury

To explore further the mechanisms by which estradiolincreases the number of newborn cells in the SVZ follow-

Fig. 4. Newly generated cells in the dorsal SVZ expressed themarker of neuronal precursor cells. Fluorescence micrographs ofBrdU-labeled cells (green) doubly stained with the early neuronalmarker PSA-NCAM (red, A–C), mature neuronal marker NeuN (red,

D–F), or astrocyte marker GFAP (red, G–I). Confocal microscopicanalysis showed that many BrdU-labeled cells coexpressed PSA-NCAM, but not NeuN or GFAP. CC, corpus callosum; STR striatum.Scale bar � 100 �m.



ing stroke-like injury, we tested whether estradiol treat-ment reduces the number of cells undergoing apoptosis.We have previously shown that estradiol inhibits neuro-nal apoptosis in the cortex of rats (Dubal et al., 1998,2001). In contrast to its early antiapoptotic actions, estra-diol did not modulate the number of TUNEL-positive cellsin the dorsal SVZ at 96 hours after the onset ischemicinjury (the number of TUNEL-positive cells; ipsilateraloil, 75.9 17.56, E2, 60.5 5.62; contralateral oil, 48.8 6.91, E2, 63.2 4.50, mean SEM of 3–5 mice per group).This demonstrates that the increase in BrdU-labeled neu-rons following estradiol treatment is not due to suppres-sion of apoptotic cell death.

Estradiol does not influence the number ofnewborn neurons at 24 hours after MCAO

To determine how soon estradiol exerts its effects onproliferation in the SVZ, we performed single (BrdU)- ordouble (BrdU/Dcx)-label immunocytochemistry at 24hours after MCAO. In contrast to the case at 96 hours, atthis earlier time point (24 hours) estradiol did not amplifythe number of BrdU-labeled cells on either the ipsi- or thecontralateral side of the SVZ or in sham-operated animals(Fig. 5A). In addition, the numbers of BrdU-positive cellsin the sham group at 24 hours were comparable to those at96 hours (Figs. 3A, 5A), suggesting that repetitive BrdUinjections over the course of 96 hours did not affect theoverall proliferating cell counts. Compared with the caseat 96 hours, equivalent proportions of BrdU-labeled cellswere colabeled with Dcx (ipsilateral oil, 64.01% 1.669%,E2, 62.13% 4.860%; contralateral oil, 69.15% 4.322%,E2, 67.23% 3.022%), suggesting that a proportion ofnewborn cells destined to differentiate into neurons re-mains constant at 24 hours and 96 hours after injury.Similarly to BrdU-labeled cells, the number of BrdU/Dcxdual-labeled immature neurons did not significantlychange in response to estradiol administration at 24 hoursafter the onset of MCAO (Fig. 5B). Confocal microscopyrevealed that a large proportion of BrdU/Dcx dual-labeledcells at 24 hours consisted of round cells that did notcontain neurite-like processes (data not shown).

Estradiol no longer amplifies the number ofnewborn neurons in the SVZ in ER�

knockout (KO) or ER� KO mice at 96 hoursafter MCAO

To establish the roles played by two forms of ER, wetested whether estradiol is capable of influencing thenumber of newly born cells in the dorsal SVZ in ER� KOand ER� KO mice at 96 hours after MCAO injury. Theamplifying effect of estradiol on the number of BrdU-labeled cells was entirely abolished in ER� KO mice aswell as in the ER� KO animals (Fig. 6A). Correspondingly,estradiol failed to increase the number of BrdU/Dcx dual-labeled cells in the dorsal SVZ in both the ER� KO and theER� KO animals (Fig. 6B). It is intriguing to note that, inestradiol-treated animals, the overall proliferative cellnumber on the injured side of the SVZ was significantlylower in both ER� KO (*P 0.05, n � 6–8) and ER� KO(*P 0.05, n � 5–6) mice compared with wild-type ani-mals (WT; Table 1). Similarly, the number of BrdU/Dcxdual-labeled cells was marginally decreased in both ER�and ER� KO animals (Table 1). The proportions of BrdU-labeled cells that coexpressed the early neuronal marker

Dcx were comparable to those of WT animals (ER� KO oil,59.02% 10.09%, E2, 73.46% 8.253%; ER� KO oil,78.56% 3.368%, E2, 72.61% 7.282%), suggesting thatthe rate of neuronal differentiation in the SVZ does notchange in the absence of either form of ER.

Because the presence of both forms of ER is crucial forestradiol to expand neuronal populations in the SVZ inresponse to injury, we performed ER� and ER� immuno-cytochemistry to understand better the mechanisms ofestradiol actions. The forebrain distributions of both ER�and ER� immunoreactivity were in strong agreementwith previous studies for mRNA (Shughrue et al., 1997)and protein (Shughrue and Merchenthaler, 2001; data notshown). However, no detectable levels of ER� or ER�immunoreactivity were observed in the SVZ of the injured(Fig. 7) or the sham-operated mice (data not shown), de-spite the use of several different doses of estradiol, assess-ment at multiple time points, and incorporation of severalsteps in the immunocytochemical procedures to optimizevisualization of low-expression antigens. These results are

Fig. 5. Estradiol did not enhance cell proliferation and/or neuro-genesis in the dorsal SVZ at 24 hours after MCAO. Estradiol did notinfluence the number of BrdU� proliferating cells in the SVZ ofovariectomized mice at 24 hours following MCAO injury or shamoperation (A). Pretreatment with estradiol did not enhance the num-ber of newborn neurons (BrdU�/Dcx�) in the SVZ in injured animals(B). The numbers represent the mean of cells counted in one section ofthe brain per animal. In both series, data represent the mean SEMof six or seven animals per group.



consistent with previous observations demonstrating thelack of ER expression in the mouse SVZ at the levels ofboth mRNA (Shughrue et al., 1997) and protein (Shugh-rue and Merchenthaler, 2001; Mitra et al., 2003; Merch-

enthaler et al., 2004) and suggest that estradiol’s ability toinfluence the number of newborn neurons in the SVZfollowing injury is indirect.

DISCUSSION

Our study clearly demonstrates that low, physiologicallevels of estradiol increase the number of newborn neu-rons in the SVZ following cerebral ischemic injury. In thisstudy, we have made three fundamental discoveries re-garding the circumstances and mechanisms of estradiolactions on proliferating cells of the SVZ. First, low physi-ological concentrations of estradiol given to ovariecto-mized mice increase the number of proliferating cells inthe SVZ after stroke-like injury without influencing therate of apoptotic cell death. This effect of estradiol isconfined to the dorsal region, the site from which neuro-blasts normally migrate to the olfactory bulb alongside therostral migratory stream, and is absent from the ventralportion of the SVZ. Moreover, this low dose of estradioldoes not exert parallel actions in the absence of injury,when uncontrolled proliferation may not be desirable. Sec-ond, on both the ipsilateral (injured) and the contralateral(noninjured) sides of the SVZ, estradiol exclusively in-creases the number of newborn cells that are destined tobecome neurons and does not influence gliosis. Third, wedemonstrate unequivocally that both ER� and ER� playessential functional roles and that the presence of bothreceptor forms is a prerequisite for the ability of estradiolto increase the number of newborn neurons in the SVZ inan animal model of stroke.

Estradiol increased the number of newborn neurons inthe SVZ at 96 hours after the onset of MCAO. Theseneuronal precursors exclusively expressed Dcx, a markerfor neuroblasts and migrating neuronal precursor cells,but did not express NeuN, a marker of mature neurons.Recently, Couillard-Despres et al. (2005) demonstratedthat Dcx is expressed in neuronal precursors only aftertheir neuronal determination and not in multipotent neu-ronal precursor/stem cells, suggesting that Dcx is a reli-able and specific marker for adult neurogenesis. Undernormal physiological conditions, the SVZ-derived neuro-blasts migrate to the olfactory bulb, where they differen-tiate into functional olfactory interneurons (Lois andAlvarez-Buylla, 1994). However, various neurodegenera-tive conditions induced by cortical aspiration, traumaticbrain injury, or cerebral ischemia redirect the migration ofneuroblasts from their normal route toward the sites ofinjury, including the cortex and striatum (Arvidsson et al.,2002; Jin et al., 2003; Goings et al., 2004; Zhang et al.,2004; Ramaswamy et al., 2005; Sundholm-Peters et al,2005; Tonchev et al., 2005). It has been well establishedthat neuroblasts in the SVZ migrate toward the boundaryzone of the ischemic lesion in rodent models of cerebralischemia (Arvidsson et al., 2002; Zhang et al., 2002, 2004,2005; Jin et al., 2003; Goings et al., 2004). Once at theischemic boundary, these cells start to exhibit multipleprocesses and ramifications (Arvidsson et al., 2002; Jin etal., 2003; Zhang et al., 2004). Recently, Yamashita et al.(2006) demonstrated that a proportion of the SVZ-derivedneuroblasts differentiates into mature neurons and startsto express NeuN at the sites of injury, where they begin tobe incorporated into the existing neuronal circuit by form-ing synapses with neighboring cells. Thus, the SVZ is animportant endogenous source of neuroblasts that are ca-

TABLE 1. Reduced Cell Proliferation and Neurogenesis in Estradiol-Treated ER� KO and ER� KO Mice at 96 Hours After MCAO1

WT(%)

ER� KO(%)

ER� KO(%)

BrdU� cells 100 19.03 53.39 5.08* 56.55 9.25*BrdU�/Dcx� cells 100 24.41 54.15 7.65 58.01 12.02

1At 96 hours after the onset of MCAO-induced injury, the number of BrdU-labeled cellsin the dorsal SVZ on the injured side was significantly lower in both ER� KO (*P 0.05,n � 6-8) and ER� KO mice (*P 0.05, n � 5-6) compared with WT animals. Similarly,the number of BrdU/Dcx dual-labeled cells was marginally decreased in both the ER�and ER� KO animals. Data are shown as percentages of WT and represent the mean SEM of five to eight animals per group.

Fig. 6. Estradiol did not regulate cell proliferation and/or neurogen-esis in the dorsal SVZ in ER� knockout (KO) or ER� KO mice at 96 hoursafter MCAO. The effect of estradiol on the number of BrdU-labeled cellsfollowing ischemic injury was abolished in the ER� KO and ER� KOanimals (A). The effect of estradiol on neurogenesis in response to MCAOwas eradicated in ER null mice. Estradiol did not increase the number ofnewborn neurons (BrdU�/Dcx�) in either the ER� KO or the ER� KOanimals (B). The numbers represent the mean of cells counted in onesection of the brain per animal. In all series, data represent the mean SEM of five to eight animals per group.



pable of migrating to the sites of injury potentially toreplace damaged neurons in response to cerebral isch-emia. However, even though the SVZ is a promising ther-apeutic target for neuronal replacement therapy, thenumbers of newborn neurons that migrate to the sites ofinjury are believed to be too small for functional recoveryof the brain (Arvidsson et al., 2002; Yamashita et al.,2006). Therefore, identification of an endogenous factorsuch as estradiol that stimulates neurogenesis in the faceof brain insults, as well as understanding the underlyingmechanisms of its action, may lead to the development ofneuronal restorative therapies against neurodegenerativediseases and injury. Recently, pharmacological therapiesdesigned to enhance endogenous neurogenesis led to func-tional recovery after stroke injury (Shyu et al., 2004;Wang et al., 2004). Whether estradiol, in addition to en-hancing neurogenesis, promotes migration of the SVZ-derived neuroblasts to the site of injury and enhancesdifferentiation/survival at the destination, leading to thestructural as well as the functional recoveries of the brain,has yet to be determined.

It is important to emphasize that we have discovered adose of estradiol that does not increase neurogenesis inthe SVZ in the absence of injury, when uncontrolled pro-liferation may be undesirable. Therefore, cell death in-duced by cerebral ischemic injury creates a neurodegen-erative microenvironment that enables low physiologicallevels of estradiol to increase the number of newbornneurons in this important neurogenic region. The en-hanced neurogenic response following estradiol treatmentis desirable in the face of injury and subsequent neuronalloss, insofar as it may enable the brain to facilitate thereplacement of injured neurons and recover function.

We also observed that, in the absence of estradiol treat-ment, stroke-like injury significantly reduced the numberof proliferating cells in the SVZ. Our results agree withthe findings of Goings et al. (2002) for mice; the authorsreported that the number of BrdU-labeled cells decreasesin response to cortical lesions, but this contrasts with theirprevious report demonstrating that, in rats, the sameinjury model increases the number of cells in the SVZ(Goings et al., 2002). It appears that the proliferativeresponse in the SVZ not only varies among species but alsodepends on the types of injury or disease. For instance,neurodegenerative conditions associated with 1)6-hydroxydopamine model of Parkinson’s disease (Bakeret al., 2004), 2) selective cholinergic system lesions(Cooper-Kuhn et al., 2004), or 3) amyloid-� peptide-induced neurotoxicity (Haughey et al., 2002) decrease pro-liferation rate in the adult rodent SVZ. Exploring theunderlying mechanisms responsible for these differencesmay provide a better understanding of how different fac-tors mediate the communication between neurodegenera-tive conditions created by brain insults, and neurogenicresponse in the SVZ.

The number of BrdU-labeled newborn cells in the SVZ isthe result of equilibrium between proliferation and celldeath. Previously we have shown that estradiol decreasesthe extent of ischemic injury by suppressing neuronalapoptosis in the cortex. Thus, we explored whether estra-diol continued to exert these antiapoptotic actions in theSVZ following stroke injury. We found that estradiol doesnot influence the number of TUNEL-positive cells in theSVZ at 96 hours after the onset of MCAO, a time point atwhich we observed the effect of estradiol on neurogenesis,

Fig. 7. ER� (A) and ER� (B) immunoreactivity was absent in theSVZ of estradiol-treated ovariectomized female mice at 96 hours afterMCAO injury. In contrast, both ER� (A) and ER� (B) immunoreac-tivities were present at high levels in other forebrain areas, includingthe bed nucleus of the stria terminalis (BNST). SVZ, subventricularzone. Scale bars � 50 �m.



indicating that an increase in the number of newbornneurons following estradiol treatment is not secondary toenhanced cell survival. Therefore, estradiol stimulates theexpansion of neuronal populations in the adult SVZ byenhancing the rate of proliferation, although the possibil-ity still exists that it also does so by influencing the rate ofnormal rostral migration of neuroblasts along the RMS.

We have begun to decipher some of the essential mech-anistic elements that contribute to estradiol’s ability toinfluence the number of newborn neurons in the face of aneurodegenerative stimulus. Our results clearly revealthat both ER� and ER� are essential mediators, and theselective ablation of one form of ER completely preventsestradiol’s ability to modulate the number of immatureneurons in the SVZ, an observation not expected if eachreceptor form plays an identical role. Thus, the results ofthe current study suggest that each receptor subtype ex-erts differential functional roles in modulating the num-ber of newborn neurons. ER� is known to exert prolifera-tive actions in numerous tissue types, including thebreast, whereas ER� plays a critical role in the organiza-tion of cortical neurons by influencing the number of cellsin the SVZ during development (Wang et al., 2003). Al-though precise roles for each ER forms have yet to bedetermined, the present study demonstrates that thepresence of both receptors is important in expansion ofneuronal populations in the SVZ after ischemic injury.The necessity of both receptor subtypes is in contrast toour previous observations demonstrating that ER� only,and not ER�, is essential to attenuate neuronal cell deathand the evolution of infarct volume after MCAO-inducedinjury (Dubal et al., 1998, 2001). Despite their essentialroles, we did not detect either ER form in the adult mouseSVZ. This agrees with previous observations demonstrat-ing the lack of ER expression in the intact as well as in theovariectomized adult mouse SVZ at the levels of mRNA(Shughrue et al., 1997) and protein (Shughrue and Mer-chenthaler, 2001; Mitra et al., 2003; Merchenthaler et al.,2004). Taken together, these findings suggest that estra-diol increases the number of newborn neurons in the adultSVZ through indirect mechanisms that require receptorslocated at distant sites, rather than through direct actionson gene expression within the newborn neurons in thisneurogenic region.

In summary, our results show that low physiologicallevels of estradiol treatment increase the number of new-born neurons in the dorsal SVZ of the adult mouse in ananimal model of stroke and that both ER� and ER� areessential mediators. The actions of estradiol are restrictedto cells that are likely to differentiate into mature neuronsand do not influence astrocytes or microglia. Togetherwith our previous findings, the results of the present studydemonstrate that low physiological doses of 17�-estradiolnot only reduce the extent of cell death but also enhanceneurogenesis following ischemic brain injury. These re-sults add yet another dimension to the diverse mecha-nisms that this pleiotropic hormone uses to maintain nor-mal brain function, attenuate the extent of injury, anddiminish deleterious changes associated with neurodegen-erative disease and injury.

LITERATURE CITED

Abercrombie M. 1946. Estimation of nuclear population from microtomesections. Anat Rec 94:239–247.

Alvarez-Buylla A, Garcia-Verdugo JM. 2002. Neurogenesis in adult sub-ventricular zone. J Neurosci 22:629–634.

Arvidsson A, Kokaia Z, Lindvall O. 2001. N-methyl-D-aspartate receptor-mediated increase of neurogenesis in adult rat dentate gyrus followingstroke. Eur J Neurosci 14:10–18.

Arvidsson A, Collin T, Kirik D, Kokaia Z, Lindvall O. 2002. Neuronalreplacement from endogenous precursors in the adult brain afterstroke. Nat Med 8:963–970.

Baker SA, Baker KA, Hagg T. 2004. Dopaminergic nigrostriatal projectionsregulate neural precursor proliferation in the adult mouse subventricu-lar zone. Eur J Neurosci 20:575–579.

Behl C. 2002. Oestrogen as a neuroprotective hormone. Nat Rev Neurosci3:433–442.

Brown JP, Couillard-Despres S, Cooper-Kuhn CM, Winkler J, Aigner L,Kuhn HG. 2003. Transient expression of doublecortin during adultneurogenesis. J Comp Neurol 467:1–10.

Cooper-Kuhn CM, Winkler J, Kuhn HG. 2004. Decreased neurogenesisafter cholinergic forebrain lesion in the adult rat. J Neurosci Res77:155–165.

Couillard-Despres S, Winner B, Schaubeck S, Aigner R, Vroemen M,Weidner N, Bogdahn U, Winkler J, Kuhn HG, Aigner L. 2005. Dou-blecortin expression levels in adult brain reflect neurogenesis. EurJ Neurosci 21:1–14.

Couse JF, Curtis SW, Washburn TF, Lindzey J, Golding TS, Lubahn DB,Smithies O, Korach KS. 1995. Analysis of transcription and estrogeninsensitivity in the female mouse after targeted disruption of the es-trogen receptor gene. Mol Endocrinol 9:1441–1454.

Dubal DB, Kashon ML, Pettigrew LC, Ren JM, Finklestein SP, Rau SW,Wise PM. 1998. Estradiol protects against ischemic injury. J CerebBlood Flow Metab 18:1253–1258.

Dubal DB, Zhu B, Yu B, Rau SW, Shughrue PJ, Merchenthaler I, KindyMS, Wise PM. 2001. Estrogen receptor-�, not -�, is a critical link inestradiol-mediated protection against brain injury. Proc Natl Acad SciU S A 98:1952–1957.

Farrar CE, Huang CS, Clarke SG, Houser CR. 2005. Increased cell prolif-eration and granule cell number in the dentate gyrus of protein repair-deficient mice. J Comp Neurol 493:524–537.

Gage FH. 2002. Neurogenesis in the adult brain. J Neurosci 22:612–613.Gerhold LM, Sellix MT, Freeman ME. 2002. Antagonism of vasoactive

intestinal peptide mRNA in the suprachiasmatic nucleus disrupts therhythm of FRAs expression in neuroendocrine dopaminergic neurons.J Comp Neurol 450:135–143.

Goings GE, Wibisono BL, Szele FG. 2002. Cerebral cortex lesions decreasethe number of bromodeoxyuridine-positive sbuventricular zone cells inmice. Neurosci Lett 329:161–164.

Goings GE, Sahni V, Szele FG. 2004. Migration patterns of subventricularzone cells in adult mice change after cerebral cortex injury. Brain Res996:213–226.

Gross CG. 2000. Neurogenesis in the adult brain: death of a dogma. NatRev Neurosci 1:67–73.

Gustafsson E, Lindvall O, Kokaia Z. 2003. Intraventricular infusion oftrkB-Fc fusion protein promotes ischemia-induced neurogenesis inadult rat dentate gyrus. Stroke 34:2710–2715.

Haughey NJ, Liu D, Nath A, Borchard AC, Mattson MP. 2002. Disruptionof neurogenesis in the subventricular zone of adult mice, and in humancortical neuronal precursor cells in culture, by amyloid beta-peptide:implications for the pathogenesis of Alzheimer’s disease. NeuromolMed 1:125–135.

Jin K, Minami M, Lan JQ, Mao XO, Batteur S, Simon RP, Greenberg DA.2001. Neurogenesis in dentate subgranular zone and rostral subven-tricular zone after focal cerebral ischemia in the rat. Proc Natl Acad SciU S A 98:4710–4715.

Jin K, Sun Y, Xie L, Peel A, Mao XO, Batteur S, Greenberg DA. 2003.Directed migration of neuronal precursors into the ischemic cerebral;cortex and striatum. Mol Cell Neurosci 24:171–189.

Komitova M, Perfilieva E, Mattsson B, Eriksson P, Johansson BB. 2002.Effects of cortical ischemia and postischemic environmental enrich-ment on hippocampal cell genesis and differentiation in the adult rat.J Cereb Blood Flow Metab 22:852–860.

Lois C, Alvarez-Buylla A. 1994. Long-distance neuronal migration in theadult mammalian brain. Science 264:1145–1148.

McEwen BS, Alves SE. 1999. Estrogen actions in the central nervoussystem. Endocrine Rev 20:279–307.

Merchenthaler I, Lane MV, Numan S, Dellovade TL. 2004. Distribution ofestrogen receptor alpha and beta in the mouse central nervous system:



in vivo autoradiographic and immunocytochemical analyses. J CompNeurol 473:270–291.

Mitra SW, Hoskin E, Yudkovitz J, Pear L, Wilkinson HA, Hayashi S, PfaffDW, Ogawa S, Rohrer SP, Schaeffer JM, McEwen BS, Alves SE. 2003.Immunolocalization of estrogen receptor beta in the mouse brain: com-parison with estrogen receptor alpha. Endocrinology 144:2055–2067.

Paxinos G, Franklin KBG. 2001. The mouse brain in stereotaxic coordi-nates, 2nd ed. San Diego: Academic Press.

Ramaswamy S, Goings GE, Soderstrom KE, Szele FG, Kozlowski DA.2005. Cellular proliferation and migration following a controlled corti-cal impact in the mouse. Brain Res 1053:38–53.

Rapp SR, Espeland MA, Shumaker SA, Henderson VW, Brunner RL,Manson JE, Gass ML, Stefanick ML, Lane DS, Hays J, Johnson KC,Coker LH, Dailey M, Bowen D, WHIMS Investigators. 2003. Effect ofestrogen plus progestin on global cognitive function in postmenopausalwomen: the Women’s Health Initiative Memory Study: a randomizedcontrolled trial. JAMA 289:2663–2672.

Rau SW, Dubal DB, Bottner M, Wise PM. 2003a. Estradiol differentiallyregulates c-Fos after focal cerebral ischemia. J Neurosci 23:10487–10494.

Rau SW, Dubal DB, Bottner MB, Gerhold LM, Guttman R, Wise PM.2003b. Estradiol attenuates markers of programmed cell death afterfocal cerebral ischemia. J Neurosci 23:11420–11426.

Shughrue PJ, Merchenthaler I. 2001. Distribution of estrogen receptorbeta immunoreactivity in the rat central nervous system. J CompNeurol 436:64–81.

Shughrue PJ, Lane MV, Merchenthaler I. 1997. Comparative distributionof estrogen receptor-� and -� mRNA in the rat central nervous system.J Comp Neurol 388:507–525.

Shumaker SA, Legault C, Rapp SR, Thal L, Wallace RB, Ockene JK,Hendrix SL, Jones BN 3rd, Assaf AR, Jackson RD, Kotchen JM,Wassertheil-Smoller S, Wactawski-Wende J, WHIMS Investigators.2003. Estrogen plus progestin and the incidence of dementia and mildcognitive impairment in postmenopausal women: the Women’s HealthInitiative Memory Study: a randomized controlled trial. JAMA 289:2651–2662.

Shumaker SA, Legault C, Kuller L, Rapp SR, Thal L, Lane DS, Fillit H,Stefanick ML, Hendrix SL, Lewis CE, Masaki K, Coker LH, Women’sHealth Initiative Memory Study. 2004. Conjugated equine estrogensand incidence of probable dementia and mild cognitive impairment inpostmenopausal women: Women’s Health Initiative Memory Study.JAMA 291:2947–2958.

Shyu WC, Lin SZ, Yang HI, Tzeng YS, Pang CY, Yen PS, Li H. 2004.Functional recovery of stroke rats induced by granulocyte colony-stimulating factor-stimulated stem cells. Circulation 110:1847–1854.

Smith MT, Pencea V, Wang Z, Luskin MB, Insel TR. 2001. Increasednumber of BrdU-labeled neurons in the rostral migratory stream of theestrous prairie vole. Horm Behav 39:11–21.

Sundholm-Peters NL, Yang HK, Goings GE, Walker AS, Szele FG. 2005.Subventricular zone neuroblasts emigrate toward cortical lesions.J Neuropathol Exp Neurol 64:1089–1100.

Suzuki S, Handa RJ. 2005. Estrogen receptor-beta, but not estrogen

receptor-alpha, is expressed in prolactin neurons of the female ratparaventricular and supraoptic nuclei: comparison with other neu-ropeptides. J Comp Neurol 484:28–42.

Tanapat P, Hastings NB, Reeves AJ, Gould E. 1999. Estrogen stimulates atransient increase in the number of new neurons in the dentate gyrusof the adult female rat. J Neurosci 19:5792–5801.

Taupin P, Gage FH. 2002. Adult neurogenesis and neural stem cells of thecentral nervous system in mammals. J Neurosci Res 69:745–749.

Tonchev AB, Yamashima T, Sawamoto K, Okano H. 2005. Enhancedproliferation of progenitor cells in the subventricular zone and limitedneuronal production in the striatum and neocortex of adult macaquemonkeys after global cerebral ischemia. J Neurosci Res 81:776–788.

Wang L, Andersson S, Warner M, Gustafsson JA. 2003. Estrogen receptor(ER)beta knockout mice reveal a role for ERbeta in migration of corticalneurons in the developing brain. Proc Natl Acad Sci U S A 100:703–708.

Wang L, Zhang Z, Wang Y, Zhang R, Chopp M. 2004. Treatment of strokewith erythropoietin enhances neurogenesis and angiogenesis and im-proves neurological function in rats. Stroke 35:1732–1737.

Wassertheil-Smoller S, Hendrix SL, Limacher M, Heiss G, Kooperberg C,Baird A, Kotchen T, Curb JD, Black H, Rossouw JE, Aragaki A, SaffordM, Stein E, Laowattana S, Mysiw WJ, WHI Investigators. 2003. Effectof estrogen plus progestin on stroke in postmenopausal women: theWomen’s Health Initiative: a randomized trial. JAMA 289:2673–2684.

Wise PM, Dubal DB, Wilson ME, Rau SW, Liu Y. 2001. Estrogens: trophicand protective factors in the adult brain. Front Neuroendocrinol 22:33–66.

Yaffe K, Sawaya G, Lieberburg I, Grady D. 1998. Estrogen therapy inpostmenopausal women: effects on cognitive function and dementia.JAMA 279:688–695.

Yamashita T, Ninomiya M, Hernandez Acosta P, Garcia-Verdugo JM,Sunabori T, Sakaguchi M, Adachi K, Kojima T, Hirota Y, Kawase T,Araki N, Abe K, Okano H, Sawamoto K. 2006. Subventricular zone-derived neuroblasts migrate and differentiate into mature neurons inthe post-stroke adult striatum. J Neurosci 26:6627–6636.

Yoshimura S, Takagi Y, Harada J, Teramoto T, Thomas SS, Waeber C,Bakowska J, Breakefield XO, Moskowitz MA. 2001. FGF-2 regulationof neurogenesis in adult hippocampus after brain injury. Proc NatlAcad Sci U S A 98:5874–5879.

Zhang RL, Zhang ZG, Zhang L, Chopp M. 2001. Proliferation and differ-entiation of progenitor cells in the cortex and the subventricular zonein the adult rat after focal cerebral ischemia. Neuroscience 105:33–41.

Zhang R, Wang Y, Zhang L, Zhang Z, Tsang W, Lu M, Zhang L, Chopp M.2002. Sildenafil (Viagra) induces neurogenesis and promotes functionalrecovery after stroke in rats. Stroke 33:2675–2680.

Zhang R, Zhang Z, Wang L, Wang Y, Gousev A, Zhang L, Ho KL, MorsheadC, Chopp M. 2004. Activated neural stem cells contribute to stroke-induced neurogenesis and neuroblast migration toward the infarctboundary in adult rats. J Cereb Blood Flow Metab 4:441–448.

Zhang RL, Zhang ZG, Chopp M. 2005. Neurogenesis in the adult ischemicbrain: generation, migration, survival, and restorative therapy. Neu-roscientist 11:408–416.



estradiol enhances neurogenesis following ischemic stroke through estrogen receptors α and β

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