17�-Estradiol Increases Astrocytic VascularEndothelial Growth Factor (VEGF) in AdultFemale Rat Hippocampus

Sharon Barouk, Tana Hintz, Ping Li, Aine M. Duffy, Neil J. MacLusky,and Helen E. Scharfman

The Nathan Kline Institute for Psychiatric Research (S.B., T.H., P.L., A.M.D., H.E.S.), Center for DementiaResearch, Orangeburg, New York 10962; Department of Biomedical Sciences (N.J.M.), OntarioVeterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1; and Departments of Childand Adolescent Psychiatry, Psychiatry, Physiology and Neuroscience (H.E.S.), New York UniversityLangone Medical Center, New York, New York 10016

Vascular endothelial growth factor (VEGF) is critical to angiogenesis and vascular permeability. It is alsoimportant in the endocrine system, in which VEGF mediates the vascular effects of estrogens in targettissues such as the uterus, a response attributed to an estrogen response element on the VEGF gene.Here we asked whether 17�-estradiol increases VEGF levels in the brain. We focused on the hippocam-pus, in which 17�-estradiol and VEGF both have important actions, and used immunocytochemistry toevaluate VEGF protein. VEGF immunoreactivity was compared in adult female rats sampled during theestrous cycle when serum levels of 17�-estradiol peak (proestrous morning) as well as when they arelow (metestrous morning). In addition, adult rats were ovariectomized and compared after treatmentwith 17�-estradiol or vehicle. The results demonstrated that VEGF immunoreactivity was increasedwhen serum levels of 17�-estradiol were elevated. Confocal microscopy showed that VEGF immuno-fluorescence was predominantly nonneuronal, often associated with astrocytes. Glial VEGF labelingwas primarily punctate rather than diffuse and labile because glial VEGF immunoreactivity was greatlyreduced if tissue sections were left in an aqueous medium overnight. We conclude that VEGF proteinin normal female hippocampus is primarily nonneuronal rather than neuronal and suggest that glialVEGF immunoreactivity has been underestimated by past studies with other methods because there isa labile extracellular pool. We suggest that estrogens may exert actions on female hippocampal struc-ture and function by increasing hippocampal VEGF. (Endocrinology 152: 1745–1751, 2011)

Vascular endothelial growth factor (VEGF) is a criticalmediator of angiogenesis and vascular permeability

(1, 2), which has been implicated in cancer and hyperten-sion. VEGF also is important in reproduction, in whichVEGF mediates, for example, estradiol-induced vascular-ization of the uterus during the ovarian cycle (3, 4). Theeffects of estradiol may be transcriptional because there isan estrogen response element on the VEGF gene (5).

In the central nervous system (CNS), VEGF mRNA isexpressed in many cells including neurons, astrocytes,and cells associated with blood vessels (6 – 8). Interest-

ingly, VEGF immunoreactivity appears to be relativelyweak in most neurons, except for select hypothalamicnuclei (9). In the hippocampus and neocortex, neuronalVEGF immunoreactivity is normally weak (8, 10), butimmunoreactivity increases after exercise and learning(11, 12), brain injury (13), hypoxia/ischemia (8, 10), orseizures (14, 15).

In this study, we asked whether 17�-estradiol in-creases VEGF protein levels in the CNS, similar to theperiphery. We focused on hippocampus in which 17�-estradiol has effects that could be mediated by VEGF

ISSN Print 0013-7227 ISSN Online 1945-7170Printed in U.S.A.Copyright © 2011 by The Endocrine Societydoi: 10.1210/en.2010-1290 Received November 7, 2010. Accepted January 25, 2011.First Published Online February 22, 2011

Abbreviations: CNS, Central nervous system; GFAP, glial-fibrillary acid protein; Ovx, ovari-ectomy; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.

B R I E F R E P O R T

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such as increased postnatal neurogenesis (16 –18),which is also increased by VEGF (19, 20). 17�-Estradioland VEGF also protect vulnerable hippocampal neu-rons in CA1 and the hilus after ischemia or seizures (15,21, 22). However, the findings have not been entirelyconsistent, possibly because of the influence of age andoxidative stress on actions of 17�-estradiol (23–25) anddistinct effects of the primary receptors for VEGF,VEGFR1 vs. VEGFR2 (26, 27).

To test whether estradiol might alter VEGF protein inhippocampus, we used adult female rats and askedwhether VEGF protein levels change in parallel withcirculating levels of 17�-estradiol during the estrouscycle. We also compared VEGF protein levels in femalerats after ovariectomy (Ovx) and treatment with 17�-estradiol or vehicle using a protocol designed to simu-late the preovulatory surge of estradiol in intact rats(28). The results support the hypothesis that 17�-estra-diol increases VEGF in the CNS: hippocampal VEGFimmunoreactivity increased in proestrous and 17�-es-tradiol-treated Ovx rats. In addition, the results showthat VEGF immunoreactivity is primarily astrocytic,not neuronal, and could easily have been missed in thepast because most hippocampal VEGF immunoreactiv-ity appears to be rapidly lost from tissue sections exvivo.

Materials and Methods

AnimalsAnimal care and use followed guidelines of the National

Institutes of Health. Adult female Sprague Dawley were sub-jects (for details, see Supplemental Materials, published onThe Endocrine Society’s Journals Online web site at http://endo.endojournals.org).

Estrous cycle stage determination and RIAVaginal cytology was used to determine cycle stage, as de-

scribed elsewhere (29). Animals were used after demonstrating atleast two 4-d estrous cycles. Estradiol concentrations were as-sessed from serum samples, as described previously (29).

Ovx and 17�-estradiol treatmentRats were ovariectomized under ketamine (80 mg/kg) and

xylazine (6 mg/kg) anesthesia as described previously (28). Es-tradiol treatment followed a previously validated injection pro-tocol, simulating the preovulatory surge (28). Three sequentialinjections were made starting at 0830–0900 h: 3 �g/kg estradiolbenzoate (sc); 11–12 h later, 4 �g/kg estradiol benzoate (sc); and12 h later, 3 �g/kg 17�-estradiol (sc). Animals were killed ap-proximately 2 h after the final injection, when hormone levelswere maximal. Vehicle-treated rats received corn oil instead of17�-estradiol.

Perfusion and immunocytochemistryDetails of procedures for immunocytochemistry using non-

confocal methods are described elsewhere (30). For confocalmicroscopy, sections were placed in PBS (3 � 10 min), followedby 1� saline sodium citrate (0.15 M NaCl per 15 mM sodiumcitrate; 95 C, 5 min), washed in PBS (3 � 5 min), blocked in 5%donkey serum in 0.3% Triton X-100 in PBS (1–2 h), incubatedin the same goat polyclonal antibody to VEGF (1:100) as non-confocal experiments, and a mouse monoclonal antibody to gli-al-fibrillary acid protein (GFAP; 1:200; Chemicon, Billerica,MA) or mouse monoclonal antibody to a neuronal nuclear an-tigen (NeuN, 1:100; Chemicon). Incubation in primary antibod-ies at 4 C overnight was followed by PBS (3 � 10 min) andincubation overnight at 4 C in secondary antibodies (fluoresceinisothiocyanate conjugated antigoat for VEGF, 1:200) or rhod-amine-conjugated antimouse for GFAP or NeuN (1:200). Im-munolabeling was absent when sections were processed withoutprimary antibody. Microscopy used a Zeiss 510 Meta confocalmicroscope (Carl Zeiss Microimaging GmbH, Jena, Germany).

Data analysisQuantification of VEGF immunoreactivity was conducted

using ImagePro software (Media Cybernetics, Bethesda, MD).Before analysis, sections were digitally photographed using thesame microscope (BX-51; Olympus America, Hauppauge, NY),camera (Spot camera model 2.2.1; Diagnostic Instruments, Ster-ling Heights, MI) and light settings. Because VEGF immunore-activity was almost exclusively punctate, immunoreactivity wasquantified as number of punctae per 100- � 150-�m rectangularviewing frame placed at random within the lamina of interest(DG: hilus, granule cell layer, molecular layer; CA1: stratumlacunosum-moleculare, radiatum, pyramidale, oriens, andoriens/alveus border of subfield CA1b; CA3: stratum pyrami-dale, lucidum, and radiatum of subfield CA3b). One hundredmicrometers was chosen because the thinnest lamina in hip-pocampus was 100 �m thick in our tissue sections. The 150-�mlength was chosen because of the curvature of some layers, mak-ing a larger rectangle not feasible to position identically through-out. Pilot studies demonstrated that it was a sufficient size tocapture the range of values for that layer (the variance for valuesusing this size frame was � 5%).

Computerized thresholding was used to ensure that VEGFimmunoreactivity was included, and background, which wasmuch lighter, was excluded. Gray-scale values computed foreach section in its entirety revealed two nonoverlappingGaussian distributions, one with relatively high values repre-senting dark VEGF immunoreactivity and one with low valuesrepresenting lighter, background staining. The threshold forquantification was placed between these two distributions.The mean gray-scale values of VEGF-immunoreactive punc-tae were similar (150 –170 with a 0 –255 scale) for all sections.In addition, the area of the viewing frame that was suprathresh-old was calculated (Supplemental Fig. 1). Septotemporal differ-ences in immunoreactivity were not evident qualitatively, but toensure that they would not influence the analysis, only dorsalhippocampus was analyzed [corresponding to 2.0–2.5 mm pos-terior to Bregma (31)].

Results are reported as mean � SEM. Statistical analysis of theVEGF data were performed using Bartlett’s test for homogeneityof variance, followed by two-way ANOVA. Because significantinteraction was observed between the main treatment effects and

1746 Barouk et al. Estradiol Increases VEGF in Hippocampus Endocrinology, May 2011, 152(5):1745–1751

regional effects (regions reflecting hippocampal layers), compar-ison of individual group means was carried out using Student’st test (P � 0.05, two tailed).

Results

VEGF immunolabeling is localized primarily toastrocytes in female rat hippocampus

Confocal microscopy demonstrated that VEGF immu-nofluorescence was primarily associated with GFAP-la-beled processes, in which it typically exhibited a punctateappearance, i.e. the strongest immunoreactivity was evi-dent as small, irregular-shaped profiles (Fig. 1A). In ad-dition, some lighter, diffuse VEGF immunolabeling of thecytoplasm of GFAP-immunoreactive cells was present(Fig. 1A). Some of the punctae appeared to be extracellu-lar, at astrocytic plasma membranes (Fig. 1B). VEGF la-beling was also in glia surrounding vessels (Fig. 1C) but didnot double label with the neuronal marker NeuN (Fig. 1,D and E). This negative result is unlikely to reflect a tech-nical difficulty because double labeling was robust with

the same methods after seizures [Supplemental Fig. 2; seealso Nicoletti et al. (15)].

Within the hilus and stratum lacunosum-moleculare,there was strong VEGF immunofluorescence. This wasalso observed using nonconfocal microscopy (Fig. 2). Inthe hilus, GFAP-labeled cells with the morphology of ra-dial glia-like progenitors were double labeled by VEGF(Supplemental Fig. 3).

Estradiol increases VEGF protein immunoreactivityin hippocampus

Quantitative comparisons of VEGF immunoreactivitywere made in rats that were euthanized midmorning(0930–1130 h) on proestrus (aged 127.0 � 7.4 d, range90–143; n � 7) or midmorning of metestrus (aged 99.9 �21.8 d, range 70–188; n � 7), which were not differentages (Student’s t test, P � 0.5406). Ovx rats treated with17�-estradiol (76.0 � 2.4 d old, range 64–86; n � 9) orvehicle (aged 74.7 � 4.5 d old, range 60–86; n � 7) werealso not different in age (Student’s t test, P � 0.7812).There was no difference in delays between Ovx and treat-

FIG. 1. Hippocampal VEGF immunoreactivity in hippocampus is associated with astrocytes. A, VEGF immunofluorescence (green) and GFAPimmunolabeling (red) of the same section is shown separately and merged (yellow; far right panel). VEGF labeling is primarily punctate stainingthat is localized to astrocytes marked by GFAP. Arrows point to the same areas of each panel. Calibration, 20 �m. B, VEGF immunofluorescence(green) is shown independent (left panel) of GFAP labeling (red; center panel), and a merged image shows double labeling (yellow; far right panel).There are two VEGF punctae (arrows) that appear to be juxtaposed to GFAP-labeled processes, possibly reflecting VEGF that is bound to astrocyticVEGF receptors. Calibration (A), 5 �m. C, VEGF and GFAP double labeling around a cross-section through a vessel shows double labeling (arrows)of glia that are likely to be associated with the blood-brain barrier. Calibration (A), 10 �m. D and E, VEGF immunofluorescence (green,arrows) was not localized to neurons labeled with the neuronal marker NeuN (red). GCL, Dentate gyrus granule cell layer. Calibration (A),50 �m (D), 20 �m (E).

Endocrinology, May 2011, 152(5):1745–1751 endo.endojournals.org 1747

ment (Ovx � 17�-estradiol, 16.4 � 0.7 d, n � 9; Ovx �vehicle, 15.8 � 0.9 d, n � 6; Student’s t test, P � 0.6598).

Serum concentrations of 17�-estradiol immediately be-fore perfusion fixation confirmed low levels in Ovx ratstreated with vehicle (3.88 � 0.944 pg/ml, n � 4) andproestrous levels in 17�-estradiol-treated rats (51.38 �5.83 pg/ml, range 38.0–65.7; n � 4; Student’s t test, P �0.000197 (29, 32).

VEGF immunoreactivity was increased in intact rats onproestrous morning compared with intact rats on me-testrous morning; there also was greater immunoreactiv-ity in Ovx rats treated with 17�-estradiol relative to ve-hicle (Fig. 2). Notably, processing sections after leavingthem in buffer overnight, rather than processing immedi-ately, led to greatly reduced immunoreactivity (Supple-mental Fig. 4). Prolonging the reaction did not restoreimmunoreactivity but led to increased background, andcell layers appeared to develop reactivity also (Supplemen-tal Fig. 4), which is important because it could lead to theimpression that neuronal expression is robust. Therefore,when delays occur or washing tissue is part of the proce-dure (e.g. for Western blot and ELISA), glial VEGF may beunderestimated. These data, in addition to those describedabove showing VEGF-labeled punctae were often juxta-posed to GFAP-labeled processes, and the identification ofglial VEGFRs (26, 27), suggest that the punctate VEGF

immunoreactivity was extracellular VEGF bound to glialVEGF receptors.

Discussion

The results show that estradiol increases VEGF proteinimmunolabeling in the hippocampus of the adult femalerat. VEGF protein appeared to be primarily astrocytic andrelatively labile.

The results are consistent with the ability of estrogensto increase VEGF synthesis (5). However, one cannot ex-clude the possibility that the increase in hippocampalVEGF immunolabeling by estradiol was due to an indirecteffect via increased neuronal activity, which has been ar-gued for other effects of estradiol (33, 34) and is importantto consider here because VEGF synthesis is activity de-pendent (11, 14, 15). In addition, estrogens can increasethe permeability of the blood-brain barrier (35–37), mak-ing it possible that peripheral VEGF could be redistribut-ing within the brain. VEGF itself could also facilitate entryof VEGF into the brain, because it increases vascularpermeability.

It is interesting that VEGF immunoreactivity was robustin stratum lacunosum-moleculare because it is a target zoneof several important afferent inputs to hippocampus. One is

FIG. 2. Increased hippocampal VEGF immunoreactivity on proestrous morning and after estradiol treatment. A, A Nissl-stained section showingthe areas of the hippocampus in which micrographs in B and C were taken. Calibration, 200 �m. B, 1, VEGF immunoreactivity (arrows) in stratumlacunosum-moleculare (SLM) and the molecular layer (MOL) from an animal that was perfused midmorning of proestrus (Pro). 2, Relatively weakVEGF immunoreactivity in an animal that was perfused midmorning of metestrus (Met) and processed concurrently with the sections from theproestrous rat (B1). V, Vessel. C, 1, In the same animal as B1, VEGF immunoreactivity (arrows) is shown in the hilus and granule cell layer (GCL). 2)Relatively weak VEGF immunoreactivity in the hilus and granule cell layer in the metestrous animal (B2). Calibration (A), 25 �m (B and C). D, ANissl-stained section is used to illustrate the locations of hippocampal areas in the bar graphs on the right, which are analyses of different strata ofthe hippocampus in intact or Ovx rats. A, Alveus; O, stratum oriens; R, stratum radiatum; LM, stratum lacunosum-moleculare; P, stratumpyramidale; G, stratum granulosum; H, hilus; M, stratum-moleculare; DG, dentate gyrus. E, Quantification of VEGF-immunoreactive punctae forcomparisons of proestrous (white, n � 7) and metestrous rats (black, n � 7). Two-way ANOVA: effect of cycle stage, F � 73.457 (df 1,132), P �0.0001; hippocampal region effect, F � 38.765 (df 10,132), P � 0.0001; interaction between cycle stage and region effects, F � 5.209 (df10,132), P � 0.0001. Asterisks indicate proestrus significantly greater than metestrus (P � 0.05). F, Quantification for 17�-estradiol-treated (white,n � 9) vs. vehicle-treated rats (black, n � 7). Two-way ANOVA: effect of 17�-estradiol treatment, F � 83.458 (df 1,153), P � 0.0001; regioneffect, F � 46.511 (df 10,153), P � 0.0001; interaction between estradiol treatment and hippocampal region effects, F � 7.800 (df 10,153), P �0.0001. Asterisks indicate a significant effect of estradiol treatment (P � 0.05).

1748 Barouk et al. Estradiol Increases VEGF in Hippocampus Endocrinology, May 2011, 152(5):1745–1751

the temporoammonic pathway from entorhinal cortex,which mediates entorhinal input to area CA1 (38). There-fore, actions of estrogens may regulate hippocampal areaCA1 via effects of VEGF on glia in this location. Stratumlacunosum-moleculare also is the site of terminals from nu-cleus reuniens of the thalamus, which has been shown toexert effects on CA1 neurons (39, 40). Interneurons are alsolocated in stratum lacunosum-moleculare, which are impor-tant in regulating area CA1 pyramidal cell activity (41). In-terestingly, stratum lacunosum-moleculare is a site of cho-linergic regulation, which has been linked to the effects ofestrogens (42).

The results have functional implications because of theknown effects of estrogens and VEGF in hippocampus, suchas stimulation of postnatal neurogenesis in the dentate gyrus(16, 18, 43). The results of the present study suggest that theeffectsofestrogensonneurogenesismaybemediated,at leastin part, by their ability to increase VEGF in the neurogenicniche. This idea is further supported by the observation thatestradiolhadarobust effectonVEGFimmunolabeling in thehilus. As mentioned above, the border of the granule celllayer and the hilus was often studded by punctate VEGFstaining on GFAP-labeled cells. Some of these cells could beradial glia-like precursors because of their immunoreactivityfor GFAP, a radial glial marker, and their morphology,which is distinct from typical hippocampal astrocytes (Sup-plemental Fig. 3).

In light of the evidence that estradiol and VEGF protecthippocampal neurons from insult or injury (15, 21, 22,44–48), the results of the present study suggest that one ofthe reasons estradiol is protective is that it increases VEGFprotein. An interesting example to consider is stroke, inwhich female rodents appear to be protected relative tomales in experimental models of stroke, and adult womenappear to be protected compared with men (48–52). Oneof the reasons for the reduced risk of stroke in femalescould be that estrogens maintain a higher level of VEGFprotein in areas of the brain such as the hippocampus.Increased VEGF may also improve the ability to recoverfrom injury because increased VEGF levels would be likelyto increase angiogenesis, which would potentially im-prove recovery (1, 53, 54). Indeed, estradiol has beenshown to prevent the down-regulation of VEGF in thebrain caused by ischemic injury in rodents (55). It is no-table that the primary site of VEGF labeling was not neu-ronal. This finding is consistent with a previous studyshowing that hippocampal VEGF mRNA in the principalcell layers of the hippocampus does not appear to changein response to 17�-estradiol in the rat (56, 57). Takentogether, it appears that both VEGF mRNA and proteinexpression in neurons of the normal adult hippocampusare not affected by 17�-estradiol. Instead, another pool of

VEGF, which appears to be associated with astrocytes, isincreased by 17�-estradiol. In contrast, after insult or in-jury, VEGF protein expression is robust in the hippocam-pal neurons (15, 43, 58), and an extracellular pool ofVEGF develops adjacent to the neuronal processes (Sup-plemental Fig. 4).

Other conditions that are known to be influenced byestrogens and have a vascular component, such as mi-graine, could also involve VEGF regulation. In migraine,many women are known to have cyclical changes in symp-toms (59, 60), and both estrogens and the vasculature havebeen implicated (61–64). Cyclical changes in VEGF pro-tein induced by estradiol could potentially contribute tocyclical variations in migraine (65).

In conclusion, our results suggest that 17�-estradiolincreases VEGF protein in the brain, analogous to actionsof 17�-estradiol in the female reproductive tract. How-ever, in brain, astrocytes are a primary target. These find-ings have implications for normal brain function becauseof the diverse roles of glia, in particular for neuroendo-crine regulation because of the contribution of astrocytesto estrogen action (66, 67), as well as for clinical condi-tions in which both estrogens and VEGF contribute.

Acknowledgments

We thank Dr. Vicky Luine for providing equipment for the RIAand Drs. Joseph Pierce and Alejandra Poveda for assistance withimage analysis.

Address all correspondence and requests for reprints to: HelenScharfman, Ph.D., The Nathan Kline Institute , 140 Old Orange-burg Road, Building 35, Orangeburg, New York 10962. E-mail:hscharfman@nki.rfmh.org or helen.scharfman@nyumc.org.

This work was supported by National Institutes of HealthGrant NS 37562, the New York State Department of Health, andthe Office of Mental Health.

Disclosure Summary: The authors have nothing to disclose.

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