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Am J Physiol Heart Circ Physiol 279: H1208-H1214, 2000;
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Vol. 279, Issue 3, H1208-H1214, September 2000

Effects of estrogen on cerebral blood flow and pial microvasculature in rabbits

M. T. Littleton-Kearney, D. M. Agnew, R. J. Traystman, and P. D. Hurn

Department of Anesthesiology and Critical Care Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We tested the hypothesis that intracarotid estrogen infusion increases cerebral blood flow (CBF) in a concentration-dependent manner and direct application of estrogen on pial arterioles yields estrogen receptor-mediated vasodilation. Rabbits of both genders were infused with estrogen via a branch of the carotid artery. Estrogen doses of 20 or 0.05 µg · ml-1 · min-1 were used to achieve supraphysiological or physiological plasma estrogen levels, respectively. CBF and cerebral vascular resistance were determined at baseline, during the infusion, and 60-min postinfusion, and effects on pial diameter were assessed via a cranial window. Pial arteriolar response to estrogen alone and to estrogen after administration of tamoxifen (10-7), an antiestrogen drug that binds to both known estrogen receptor subtypes, was tested. No gender differences were observed; therefore, data were combined for both males and females. Systemic estrogen infusion did not increase regional CBF. Estradiol dilated pial arteries only at concentrations ranging from 10-4-10-7 M (P <=  0.05). Pretreatment with tamoxifen alone had no effect on arteriolar diameter but inhibited estrogen-induced vasodilation (P < 0.001). Our data suggest that estrogen does not increase CBF under steady-state conditions in rabbits. In the pial circulation, topically applied estradiol at micromolar concentrations dilates vessels. The onset is rapid and dependent on estrogen receptor activation.

microcirculation; pial circulation; precapillary vessels; tamoxifen; cranial window


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ESTROGEN HAS RECENTLY BEEN demonstrated to have significant neuroprotective properties and may be a potential therapeutic agent in neuroinjury in females. Our laboratory and others have observed that chronically increased estrogen availability augments cerebral blood flow (CBF) during global (27, 51) and focal cerebral ischemia in rodents (1). Although estrogen has been well established as a vasodilator of coronary and other peripheral vascular beds, it remains to be shown whether the steroid alters cerebrovascular function or cerebral perfusion under physiological, nonischemic conditions. Early studies indicate that women have a higher mean CBF compared with men (24, 53, 57) and greater homogeneity in regional flow distribution (53). Presently, few data exist regarding the acute effect of estrogen on cerebral hemodynamics or on vessel diameter when applied directly to cerebral microvessels in situ. A single early report suggests that acute estrogen infusion increases CBF in rat (23). More recently, Magness and associates (38) have shown in female sheep that long-term, systemic estrogen treatment mildly enhances CBF, whereas acute steroid infusion has no effect (38). In vitro, chronic estrogen depletion augments vasoconstriction (16), whereas estrogen infusion reduces vascular tone in cerebral arteries (20). A dose-dependent effect of estrogen has been linked to cGMP signaling in hippocampal and cortical brain regions (46). Therefore, it is possible that estrogen exerts a dose-dependent effect on the cerebral microvasculature. In light of these earlier studies, the purpose of this investigation was to determine whether estrogen administered directly into the cranial circulation increases CBF or vascular diameter at physiologically relevant or supraphysiological plasma concentrations. We hypothesized that sustained intracarotid estrogen infusion increases regional CBF in a concentration-dependent manner and that direct application to pial arterioles yields a dose-dependent, estrogen receptor-mediated vasodilation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. All protocols for this study were approved by the University Animal Care and Use Committee. Adult sexually mature New Zealand White rabbits (weight 2.5-4.0 kg) were used in both the blood flow and cranial window protocols. Anesthesia was induced by marginal ear vein injection of pentobarbital sodium (20-35 mg/kg), followed by a continuous, intravenous infusion at 4 mg · kg-1 · h-1. A tracheostomy was performed, and the animal was mechanically ventilated with supplemental oxygen to maintain normal arterial blood gases. Both femoral arteries and veins were cannulated bilaterally with polypropylene catheters for drug administration (venous; PE-90) or arterial pressure monitoring and blood gas evaluation (arterial; PE-90). Arterial blood pressure was continuously recorded on a Gould-Brush polygraph in all protocols. Arterial blood gases (Ciba Corning Diagnostics, Essex, UK), oxygen content, saturation, and hemoglobin (Co-oximeter; OSM 3 Radiometer, Copenhagen, Denmark), and glucose concentration (glucose analyzer, model 27; Yellow Springs Instruments, Yellow Springs, OH) were controlled throughout the experimental protocols.

The rabbit was chosen as a model for these sets of experiments for several reasons. First, we have published data in the rabbit (27) to show that chronic estrogen replacement limits intraischemic reduction of CBF. These data suggest that estrogen has a vasorelaxant effect on the cerebral vasculature. Second, the rabbit is a well-studied species from the perspective of reproductive endocrinology. A primary advantage is that the rabbit is a reflex ovulator, i.e., basal estrogen is constant because there is a lack of cyclic variation in hormone levels over a menstrual cycle, thus eliminating the need for surgical ovariectomy. It is precisely this lack of variation that allows ease of manipulation of estrogen over a wide range of plasma levels, independent of progesterone. Finally, the rabbit brain is a reasonable model of human cerebral circulation, particularly because it lacks a rete mirable.

Cerebral blood flow studies. Rabbits were infused with Premarin (Ayerst Laboratories, Philadelphia, PA) at 20 µg · ml-1 · min-1 (males and females, n = 8 per group), 50 ng · ml-1 · min-1 (females, n = 5), or saline-infused time controls (males and females, n = 3 per group). These doses were chosen on the basis of preliminary data which indicated that the low dose provided physiologically relevant circulating plasma estrogen levels, whereas the high dose was supraphysiological. Estrogen is normally low in the unstimulated rabbit, ranging from 10-30 pg/ml in the ovulatory (45, 61) animal and rising to 40-90 pg/ml in pregnancy (45, 61). The right thorax was opened, and a left atrial catheter was inserted for injection of radiolabeled microspheres. The incision was closed, and the lung was reinflated. The right facial branch of the carotid artery was cannulated and (PE-50) then threaded retrograde toward the carotid trunk for infusion of drug or isotonic saline. The facial branch cannulation was employed to avoid altering carotid blood flow or inflow to brain but to allow high drug concentrations to be directed to the cerebral circulation.

Regional CBF was evaluated at baseline, 30 and 60 min of ongoing infusions and 1 h postinfusion using the radiolabeled microsphere technique (15 ± 0.5-µm diameter spheres) (24). Briefly, a dose of ~1.5 × 106 microspheres labeled with either 57Co, 114In, 113Sn, 95Nb, or 45Sc (New England Nuclear-DuPont, Boston, MA) was injected into the left atrium as previously described followed by a 1-ml saline flush (27). The femoral arterial reference sample was withdrawn at a rate of 1.94 ml/min. After being harvested and fixed in Formalin, the brain was dissected into the cerebellum, medulla, pons, midbrain, hippocampus, caudate nucleus, and the remaining cerebral hemispheres. Additional tissue samples were collected from the right cheek muscle and tongue as points of reference in the extracranial circulation. Radioactivity of tissue and blood reference samples were counted on a autogamma scintillation spectrometer (model 5530; Packard Instruments, Downers Grove, IL) for estimation of the number of microspheres in each tissue sample. Blood flows were determined as previously described, using the reference organ technique (27, 34). Cerebral vascular resistance (CVR) was calculated as mean arterial blood pressure (MAP; mmHg) divided by CBF (ml · min-1 · 100 g tissue-1).

Arterial blood samples were collected from the femoral artery at baseline, 30 and 60 min infusion, and 1 h postinfusion, and then centrifuged (1,500 rpm; 20 min); plasma was obtained for determination of estrogen levels using a commercially available kit (Diagnostic Products, Los Angeles, CA). The assay sensitivity is <= 8 pg/ml, with minimal cross-reactivity as previously described (27).

After the surgical preparation was completed, animals were given a 30-min equilibration time prior to the experimental protocol. Baseline arterial blood gases, plasma estrogen, and CBF were measured, and the animals were then infused with Premarin diluted in 10 ml saline to deliver either 20 µg · ml-1 · min-1 or 50 ng · ml-1 · min-1 or given isotonic saline alone in equivalent volume. Physiological measurements, estrogen level, and CBF were again measured at 30 and 60 min of infusion, then at 1 h postinfusion. Microspheres were injected immediately after blood samples were obtained. After final measurements, the animals were overdosed with pentobarbital and killed by KCl injection.

Vascular diameter studies. In these protocols, 19 pial arterioles were studied in the female (n = 8 animals, 12 vessels) and male (n = 5 animals, 7 vessels) rabbits, using a parietal cranial window and intravital microscopy, as previously described (12, 28). Briefly, a craniotomy was performed over the parietal cortex with a cooled, high-speed drill. Bone bleeding was controlled, and the site was enclosed with dental acrylic, which serves as a "well" for the superfusion fluid. The dura was excised, and the vessels were exposed. The cranial window was sealed with a glass cover slip and suffused with artificial cerebrospinal fluid (aCSF) at controlled temperature (37°C), pH, PCO2, and PO2. Inlet and outlet tubing allow infusion of experimental agents and control of intrawindow pressure (5 mmHg). Intrawindow temperature was held at 37-38°C throughout the procedure by adjustment of a heat lamp focused on the cranial window. Pial arteriolar diameter was measured (50-100 µm, baseline diameter) using a Zeiss compound microscope with fiberoptic epi-illumination interfaced to a charge-coupled device (CCD) camera, high-resolution monitor, and a Super-VHS video cassette recorder. Inner vessel diameter was measured by offline video analysis with resolution of 2 µm. In all preparations, postoperative vessel reactivity was confirmed by superfusion of acetylcholine (ACh, 10-5 M) into the cranial window and visualization of prompt vasodilation. If vessels were unresponsive to ACh, then the preparation was considered unacceptable and excluded from further study (2 animals).

In the first series of animals, we determined whether estrogen produces vasodilation when directly applied to pial arterioles. Baseline diameter was measured, then 17beta -estradiol (10-4-10-9 M) or ethanol vehicle at equiconcentration was superfused (1.25 ml/min over 5 min) into the cranial window. Order of application for the various superfusion concentrations was randomized among animals. The drug was allowed to dwell for 5 min, and the arteriole diameters were then remeasured. The window was then superfused with warmed aCSF for 5 min, and a new baseline arteriolar diameter was measured prior to infusion of the next drug concentration.

In a second animal series, we utilized tamoxifen, a competitive estrogen receptor antagonist, to determine whether the observed estrogen-induced pial arteriolar vasodilation was receptor dependent. Tamoxifen is a nonspecific estrogen antagonist, acting at both alpha - and beta -receptor subtypes. There is no receptor subtype-specific antagonist available at present. The dose of tamoxifen (10-7 M) was chosen based on previous work indicating significant antiestrogen potency at this concentration (35, 36) and for its ready solubility in aCSF. After baseline measurements, vessel response to estradiol (10-5 M) was determined. Tamoxifen in warmed aCSF was infused into the window and allowed to dwell for 30 min and then followed by a second superfusion of estradiol. The (10-5 M) estradiol concentration was chosen because it produced the most robust dilation in the previous series of animals. Pial arteriolar diameter was measured at baseline, estradiol administration, posttamoxifen, and after the second estradiol treatment.

Statistical analysis. Data are expressed as means ± SE. All repeated measurements were evaluated by two-way repeated measures analysis of variance (RMANOVA). If the F statistic for group treatment or the interaction between group and time was significant (P <=  0.05), then a one-way RMANOVA, means among individual time points were compared by the Newman-Keuls multiple comparison test.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cerebral blood flow studies. Table 1 summarizes the physiological data, including pH, PO2, PCO2, and MAP for these experiments. In our animals, baseline arterial plasma estradiol was 2.5 ± 1.8 pg/ml in females and 12.9 ± 3.7 pg/ml in males. As expected, the high- and low-dose infusions produced supraphysiological and physiological plasma levels, respectively. The high-dose estrogen infusion (20 µg · ml-1 · min-1) produced 1.5 ng/ml plasma concentrations at 60 min of infusion, which fell to 413 pg/ml (P<= 0.001) by 1 h postinfusion. Low-dose estrogen infusion (0.05 µg · ml-1 · min-1) produced a plasma concentration of 28 pg/ml at 60 min of infusion, falling to 7 pg/ml by 1 h postinfusion (P <=  0.05). Based on the steady-state plasma estrogen levels at 60 min infusion and average CBF, the brain estrogen delivery for the high-dose infusion was calculated to be 1.8 × 10-10 mol · min-1 · 100 g tissue-1. For the 0.05 infusion group, estimated estrogen delivery was 4.3 × 10-12 mol · min-1 · 100 g tissue-1.

                              
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  		Table 1.   Physiological data during sustained intracarotid estrogen infusion

Figures 1 and 2 show averaged hemispheric CBF and CVR in response to high- and low-dose estrogen infusion. Despite elevation of plasma estrogen and contrary to our expectations, regional CBF and CVR varied little over the 60 min of estrogen infusion or the 60 min after infusion cessation. Even high-dose estrogen treatment produced no change in hemispheric blood flow (Fig. 1). We also examined blood flow in other brain regions including hippocampus, hypothalamus, caudate nucleus, and brain stem and found no change in perfusion during or following estrogen infusion (data not shown). Estrogen infusion had no effect on blood flow to facial muscle or tongue, which were used as reference points to estimate the effect of the hormone on extracranial tissues supplied by the facial artery (data not shown).


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  		Fig. 1.   Effects of intracarotid infusion of physiological (0.05 µg/min; n = 6) or supraphysiological (20 µg/min; n = 15) estrogen concentrations or saline (SAL; n = 6) on hemispheric blood flow.



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  		Fig. 2.   Effects of intracarotid infusion of physiological (0.05 µg/min; n = 6) or supraphysiological (20 µg/min; n = 15) estrogen concentrations or saline (SAL; n = 6) on cerebral vascular resistance (CVR).

Cerebral microvascular response to estrogen. Arterial PO2, PCO2, pH, and MAP were constant throughout the protocol. Gender had no effect on responsiveness of the pial arterial vessels to estradiol; therefore, data from both male and female rabbits were combined. Pial venous responses were not examined in these series of studies. The pial arterial bed of the rabbits contains few precapillary vessels with diameters larger than 105 µm. Consequently, we examined medium-sized pial arterioles to more readily detect small changes in vessel diameter. In our studies of dose response, the arterial vessel diameters ranged from 54 to 96 µm. As shown in Fig. 3, 17beta -estradiol administered at low concentrations (10-8 M and 10-9 M) did not produce pial arteriolar dilation. At high concentrations (10-4-10-7 M), pial arteriolar diameter increased in response to hormone application. There was no dose dependency observed, in that all concentrations tested produced similar dilation (9 ± 1.7, 16 ± 2.5, 13 ± 2.5, and 15 ± 2.5% of baseline diameter; P < 0.05) at 10-4-10-7 M concentrations, respectively. We observed no significant change in pial arteriolar diameter at any concentration of ethanol vehicle. Figure 4 shows the effect of estradiol infusion when vessels were pretreated with tamoxifen. In these studies, baseline pial arteriole diameters ranged from 36-103 µm; mean diameter was 54 µm. Before tamoxifen, estrogen dilated pial arterioles (n = 16) in a fashion similar to that observed in the first animals series (21 ± 2.6% of baseline). Application of tamoxifen alone resulted in no change in vessel diameter (96.9 ± 5.2% baseline). All pial arterioles were unresponsive to estradiol superfusion after tamoxifen treatment (99 ± 1.5% of baseline; P < 0.001).


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  		Fig. 3.   Pial vessel dilation expressed as percentage of baseline diameter in response to 5 min of superfusion of 17beta -estradiol (n = 11 vessels) or ethanol vehicle (n = 8) vessels. Vessel diameters ranged from 54-96 µm. *P < 0.05 from baseline.



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  		Fig. 4.   Pial vessel dilation to 17beta -estradiol (10-5 M) superfusion before and 30 min after administration of tamoxifen (10-7 M). Vessels ranged from 36-102 µm. ***P < 0.001.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates three major findings. First, 17beta -estradiol produces rapid vasorelaxation when applied directly to the pial arteriolar microvasculature, suggesting that the steroid is vasoactive at high local concentrations. The rapidity with which the hormone exerts this effect (within minutes of application) strongly suggests that dilation is not mediated through classic, steroid transcriptional activity mechanisms involving interactions with nuclear receptor-estrogen response elements. Second, the antiestrogen drug tamoxifen completely blocks the estrogen vasodilatory signal in pial vessels within the window of our observations. This effect suggests that the vasodilation is receptor mediated, presumably by interaction with a membrane or cytosolic estrogen receptor. These data do not distinguish the specific subtype of estrogen receptor involved, and further experiments are needed to elucidate signaling mechanisms downstream from receptor activation. Third, acute intracarotid estrogen infusion, resulting in even supraphysiological plasma estrogen levels, does not alter CVR or CBF in the intact vascular bed in the anesthetized rabbit. Under steady-state conditions without fluctuations in cerebral perfusion pressure, the direct actions of estrogen on brain blood flow appear minimal. In light of these findings, it is unlikely that estrogen is a potent vasodilator of the cerebral circulation at the low plasma concentrations ordinarily observed in female, reproductively active animals. At high local concentrations, however, the steroid can elicit microvascular dilation.

A variety of studies indicate that ovarian hormones modulate vascular reactivity of peripheral, uterine, and coronary arteries (17, 42, 59). However, accounts of the role of estrogen as a modulator of the cerebral vasculature reactivity in vivo are limited. The lack of effect of systemic estrogen infusion that we observed in the present study mirrors that of Magness and associates (38), who noted that in conscious neutered female sheep, acute estrogen infusion produced no change in CBF. Other investigations have also revealed minimal influence of circulating plasma estrogen on steady-state CBF in either conscious or unconscious states (1, 26, 27). In the present study, neither high nor low circulating estrogen concentrations effected cerebrovascular resistance or flow. These findings are generally in accord with several earlier reports, but differ from those of Goldman et al. (23), who showed that high-dose estrogen infusion transiently (10 min) raised CBF in the rat (23). Since we employed an episodic approach to measuring CBF with the radiolabeled microsphere technique, it is conceivable that we missed an early transitory estrogen-induced fall in CVR or increase in CBF. Alternatively, a possibility exists that the absence of change in cerebral vascular tone following estrogen infusion may be related to anesthesia-induced vasomotor tone depression. However, this is unlikely since Magness et al. (38) observed similar effects of acute estrogen replacement on CBF in conscious sheep. Another more plausible explanation is that in a nonischemic, presumably autoregulating, cerebrovascular bed, temporary vasodilatory effects of estrogen have little physiological role.

Clinical studies of nonischemic CBF patterns demonstrate higher gray matter blood flow in premenopausal women compared with men (15, 54) and better cerebral vasodilatory capacity (31, 32) than their male counterparts. These clinical observations suggest that endogenously produced estrogen may modulate regional CBF. However, we noted no change in CBF or CVR following continuous intracarotid infusion of estrogen at either physiological or supraphysiological plasma estrogen levels. We studied only females at the low-dose estrogen infusion rate. However, we examined both males and females in the high-dose group and determined that the lack of effect of estrogen on CBF was irrespective of gender. Similarly, no gender effect was observed in the cranial window experiments conducted in both male and female animals.

Micromolar or higher concentrations elicited a brisk vasodilation by an estrogen receptor-dependent, nontranscriptional mechanism. We did not seek to mimic such high microvascular concentrations (e.g., 10-5 M) in the systemic treatment experiments with Premarin, so we do not know whether increased CBF would have resulted. It is possible that the capacity of estrogen to act as a cerebral vasodilator is limited to the pial circulation or to the microvasculature vs. large cerebral resistance vessels, which control total cerebrovascular resistance. Nevertheless, the present data serve as a first characterization of the vasodilatory activity of estrogen in an intact cerebral microvascular network. Previous in vitro data support this concept. Estrogen lowers myogenic tone and responsiveness to vasopressors in rat cerebral arterioles (20). Furthermore, estrogen deficiency enhances vasoconstrictor responses in rabbit basilar artery rings (16, 19). Finally, substantial data indicate that estrogen modulates noncerebral vessel tone, particularly in the coronary circulation. Incubation with 17beta -estradiol relaxes precontracted femoral, aortic, and coronary artery rings and inhibits contractile responses of coronary artery smooth muscle cells (3, 14, 18, 59). 17beta -Estradiol enhances endothelium-dependent vasodilation of precontracted, isolated arteries of ovariectomized rabbits (21) and pigs (3), as well as nonendothelium-dependent relaxation of both rat and mouse aortic rings (18).

We found estrogen-induced relaxation was neither gender nor log-dose dependent. However, it is possible that a dose-response relationship does exist but within much narrower limits than those of our study. Nevertheless, once a vasoactive concentration was attained, an apparently maximum effect was observed. Estrogen receptors are present in both endothelial and vascular smooth muscle cells (30, 52), and diverse studies demonstrate hormone-mediated vasodilation in a variety of vascular beds (38, 42), including those of the skin (2, 62), coronary (14, 59), and brain (23, 33). The rapidity of the increase in vessel diameter subsequent to estrogen superfusion clearly argues against interaction with a classic nuclear receptor and modulation of gene transcription, which is a more lengthy process (3). We observed that competitive estrogen receptor blockade with tamoxifen entirely reversed estrogen-induced vasodilation, yet did not alter vascular responsiveness to the endothelium-dependent vasodilator acetylcholine (data not shown). Therefore, tamoxifen did not produce a generalized depression of vasodilation. Estrogen likely activates a microvascular receptor and a nontranscriptional intracellular signaling mechanism in our preparation.

The exact vasodilatory mechanisms by which estrogen acts are not known, and it is not always clear which mechanisms are acting at physiologically relevant concentrations. Reports from many laboratories support the existence of rapid-acting, estrogen-stimulated, nontranscriptional modulation of vasomotor tone (9, 22, 35, 57) and initiation of intracellular signaling cascades (13). An interaction between estrogen and nitric oxide (NO) has been widely implicated in the noncerebral vasculature (8, 10, 22), possibly via nitric oxide-induced opening of calcium-activated K channels (14), and in the cerebral circulation (20, 41, 46, 51). Recent findings in cell culture suggest that estradiol triggers an intracellular calcium/calmodulin-dependent translocation of caveolae-bound endothelial nitric oxide synthase (eNOS) to the cell cytosol (10, 22, 60) that could account for a rapid fall in vasomotor tone. Alternatively, estrogen increases eicosanoid production in the vasculature, including dilatory prostacyclin (6, 11, 29, 43). In addition, estrogen can modulate interactions between eNOS and cyclooxygenase products (7), so cross talk between these vasodilator signaling systems may be involved in the mechanisms of estrogen. Finally, the vascular signaling of estrogen may involve a cytochrome P-450-dependent, endothelium-derived hyperpolarizing factor (5). Further experiments are needed to elucidate signaling mechanisms downstream from estrogen receptor activation, including key vasodilatory mediators.

In the systemic infusion experiment, we sought to determine whether estrogen increases CBF, since previous in vitro observations suggest that the steroid is an endothelium-dependent vasodilator. We observed no effect of intraluminal estrogen delivery, as assessed by evaluation of CBF during estrogen infusion. Similarly, abluminal application did not induce vasodilation in the cranial window preparation at concentrations less than 10-7 M. Although we did not measure brain or CSF accumulation of estrogen in the present study, it is likely that the carotid artery infusion also elevated brain and CSF estrogen concentrations. The capacity of the lipophilic steroid to cross the blood-brain barrier has been well studied. Data from humans (44, 56) as well as animals, including rabbits (4, 40, 47, 48, 49), demonstrate a direct relationship between plasma and brain/CSF estrogen levels. Based on these reports, it is evident that CSF estrogen levels are similar among species, and basal levels have been reported as 1.0-1.8 × 10-11 M. Pardridge and colleagues (48) demonstrated that estrogen penetration of the blood-brain barrier occurs in humans and is inversely proportional to sex hormone binding globulin (SHBG) levels. Premenopausal women demonstrate CSF estrogen concentrations that are ~2.5% of plasma levels (44). CSF estrogen concentration is similar in animals across species that possess SHBG for estradiol (39). Accumulation of estradiol across the blood-brain barrier is established very early in life in the rabbit but in adulthood in the adult rat (48, 49).

There is little evidence that CSF estrogen concentration determines the vasoreactivity of the steroid. However, CSF estrogen could be important if the hormone acts via a metabolic mechanism of vasodilation. If estrogen increases local neuronal metabolism over time, then CBF should increase to match increased oxygen utilization. The present study did not measure cerebral metabolic rate over time in these animals, so our experimental design does not address this hypothesis. Nevertheless, hormone infusion over 60 min failed to alter CBF within the window of our observations.

The cranial window experiments examined the effects of estradiol on pial arterioles without dilution in the systemic circulation and varying delivery conditions. This permitted us to determine the magnitude of concentration required to elicit pial vasodilation in situ. We noted that infusion concentrations at 10-8 or 10-9 M produced no effect on vessel diameter. This dose-response relationship was not extended to 10-11 or 10-12 M, which would have mimicked the basal CSF estrogen concentrations attained over the reproductive cycle. Although it is feasible that estrogen has a biphasic vasodilatory dose-response relationship, eliciting a vasodilation at 10-7-10-4 M that disappears at 10-9 M and 108 M and reappears at 10-11 M, this seems unlikely. It is more likely that pial vessels dilate at concentrations of 10-7 M or greater, which is supraphysiological for either plasma or CSF.

In conclusion, our data suggest that estrogen is not a potent vasodilator of the cerebral circulation under steady-state, physiological conditions. These data do not exclude a facilitatory or permissive role for estrogen in autoregulatory challenges or responses to ischemic states when perfusion pressure is compromised. In contrast, 17beta -estradiol is vasoactive at micromolar concentrations in the intact pial circulation. When pial vasodilation is evoked, the onset is rapid and dependent on estrogen receptor activation.


    ACKNOWLEDGEMENTS

This work was supported in part by National Institutes of Health Grants RO1-NS-33668 and NR-03521 (to P. D. Hurn) and KO7-NR-00072-03 (to M. T. Littleton-Kearney).


    FOOTNOTES

Address for reprint requests and other correspondence: M. Littleton-Kearney, 1508-B, 600 N. Wolfe St., Baltimore, MD 21287 (E-mail: mkearney{at}jhmi.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 26 January 2000; accepted in final form 19 April 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alkayed, NJ, Harukuni I, Kimes AS, London ED, Traystman RJ, and Hurn PD. Gender-linked brain injury in experimental stroke. Stroke 29: 159-166, 1998[Abstract/Free Full Text].

2.   Al-Khali, F, Eriksson M, Landgren BM, and Schenck-Gustafsson K. Effect of conjugated estrogen on peripheral flow-mediated vasodilation in postmenopausal women. Am J Cardiol 82: 21-218, 1998.

3.   Bell, DR, Rensberger HJ, Kortnik DR, and Koshy A. Estrogen pretreatment directly potentiates endothelium-dependent vasorelaxation of porcine coronary arteries. Am J Physiol Heart Circ Physiol 268: H377-H383, 1995[Abstract/Free Full Text].

4.   Bixo, M, Winblad WB, Selstam G, and Andersson A. Comparison between pre- and postovulatory distributions of oestradiol and progesterone in the brain of the PMSG-treated rat. Acta Physiol Scand 128: 241-246, 1986[Web of Science][Medline].

5.   Bobadilla, RA, Henkel CC, Henkel EC, Escalante B, and Hong E. Possible involvement of endothelium-derived hyperpolarizing factor in vascular responses of abdominal aorta from pregnant rats. Hypertension 30: 596-602, 1997[Abstract/Free Full Text].

6.   Bolego, C, Cignarella A, Ruzza R, Zarour C, Messi E, Zanisi M, and Puglisi L. Differential effects of low and high-dose estrogen treatments on vascular responses in female rats. Life Sci 60: 2291-2303, 1997[Web of Science][Medline].

7.   Case, J, and Davison CA. Estrogen alters relative contribution of nitric oxide and cyclooxygenase products to endothelium-dependent vasodilation. J Pharmacol Exp Ther 291: 524-530, 1999[Abstract/Free Full Text].

8.   Caulin-Glaser, T, Garcia-Cardena G, Sarrel P, Sessa WC, and Bender JR. 17beta -estradiol regulation of human endothelial cell basal nitric oxide, independent of cytosolic Ca2+ mobilization. Circ Res 81: 885-892, 1997[Abstract/Free Full Text].

9.   Chen, J, Adachi N, Liu K, and Arai T. The effects of 17 beta-estradiol on ischemia-induced neuronal damage in the gerbil hippocampus. Neuroscience 87: 817-822, 1998[Web of Science][Medline].

10.   Chen, Z, Yuhanna IS, Galcheva-Gargova Z, Karas RH, Mendelsohn ME, and Shaul PW. Estrogen receptor alpha  mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J Clin Invest 103: 401-406, 1999[Web of Science][Medline].

11.   Chang, WC, Nakao J, Orimo H, and Murota SI. Stimulation of prostacyclin biosynthetic activity by estradiol in rat aortic smooth muscle cells in culture. Biochim Biophys Acta 619: 107-118, 1980[Medline].

12.   Clavier, NM, Kirsch JR, Hurn PD, and Traystman RJ. Loss of cerebral vascular reactivity to muscarinic stimulation during post-ischemic delayed hypoperfusion. Anesthesiology 79: A586, 1993.

13.   Collins, P, and Webb C. Estrogen hits the surface. Nat Med 5: 1130-1131, 1999[Web of Science][Medline].

14.   Darkow, DJ, Lu L, and White RE. Estrogen relaxation of coronary artery smooth muscle is mediated by nitric oxide and cGMP. Am J Physiol Heart Circ Physiol 272: H2765-H2773, 1997[Abstract/Free Full Text].

15.   Davis, SM, Ackerman RH, Correia JA, Alpert NM, Chang J, Buonanno F, Kelley RE, Rosner B, and Taveras JM. Cerebral blood flow and cerebro-vascular CO2 reactivity in stroke-age normal controls. Neurology 33: 391-399, 1983[Abstract/Free Full Text].

16.   Dhall, U, Cowen T, Haven AJ, and Burnstock G. Effect of oestrogen and progesterone on noradrenergic nerves showing serotonin-like immunoreactivity in the basilar artery of the rabbit. Brain Res 442: 335-339, 1988[Web of Science][Medline].

17.   Farhat, M, Abi-Younes S, and Ramwell PW. Non-genomic effects of estrogen and the vessel wall. Biochem Pharmacol 51: 571-576, 1996[Web of Science][Medline].

18.   Freay, AD, Curtis SW, Korach KS, and Rubanyi GM. Mechanism of vascular smooth muscle relaxation by estrogen in depolarized rat and mouse aorta. Circ Res 81: 242-248, 1997[Abstract/Free Full Text].

19.   Futo, J, Shay J, Block S, Hold J, Beach M, and Moss J. Estrogen and progesterone withdrawal increases cerebral reactivity to serotonin in rabbit basilar artery. Life Sci 50: 1165-1172, 1992[Web of Science][Medline].

20.   Geary, GG, Krause DN, and Duckles SP. Estrogen reduces myogenic tone through a nitric oxide-dependent mechanism in rat cerebral arteries. Am J Physiol Heart Circ Physiol 275: H292-H300, 1998[Abstract/Free Full Text].

21.   Gisclard, V, Miller VM, and Vanhoutte PM. Effect of 17beta -estradiol on endothelium-dependent responses in the rabbit. J Pharmacol Exp Ther 244: 19-22, 1987[Abstract/Free Full Text].

22.   Goetz, RM, Thatte HS, Prabhakar P, Cho MR, Michel T, and Golan DE. Estradiol induces the calcium-dependent translocation of endothelial nitric oxide synthase. Proc Natl Acad Sci USA 96: 2788-2793, 1999[Abstract/Free Full Text].

23.   Goldman, H, Skelley EB, Sandman CA, Kastin AJ, and Murphy S. Hormones and regional brain blood flow. Pharmacol Biochem Behav 5: 165-169, 1976[Web of Science][Medline].

24.   Gur, RC, Gur RE, and Obrist WD. Sex and handedness difference in cerebral blood flow during rest and cognitive activity. Science 217: 659-661, 1982[Abstract/Free Full Text].

25.   Heymann, MA, Payne BD, Hoffman JIE, and Rudolph AM. Blood flow measurements with radionuclide-labeled particles. Prog Cardiovasc Dis 20: 55-79, 1977[Web of Science][Medline].

26.   Holshneider, DP, and Scremin OU. Effects of ovariectomy on cerebral flow in rats. Neuroendocrinology 67: 260-268, 1998[Web of Science][Medline].

27.   Hurn, PD, Littleton-Kearney MT, Kirsch JR, Dharmarajan AM, and Traystman RJ. Postischemic cerebral blood flow recovery in the female: effect of 17beta estradiol. J Cereb Blood Flow Metab 15: 666-672, 1995[Web of Science][Medline].

28.   Hurn, PD, Traystman RJ, Shoukas AA, and Jones MD. Pial microvascular hemodynamics in anemia. Am J Physiol Heart Circ Physiol 264: H1231-H1235, 1993.

29.   Jun, SS, Chen Z, Pace MC, and Shaul PW. Estrogen upregulates cyclooxygenase-1 gene expression in ovine fetal pulmonary artery endothelium. J Clin Invest 102: 176-183, 1998[Web of Science][Medline].

30.   Karas, RH, Patterson BL, and Mendelsohn MD. Human vascular smooth muscle cells contain functional estrogen receptors. Circulation 89: 1943-1950, 1994[Abstract/Free Full Text].

31.   Karnik, R, Valentin A, Winkler WB, Khaffaf N, Donath P, and Swlany J. Sex-related differences in acetazolamide induced cerebral vasomotor reactivity. Stroke 27: 56-58, 1996[Abstract/Free Full Text].

32.   Kastrup, A, Thomas C, Hartmann C, and Schabet M. Sex dependency of cerebrovascular CO2 reactivity in normal subjects. Stroke 28: 2353-2356, 1997[Abstract/Free Full Text].

33.   Kastrup, A, Li TQ, Glover GH, Kruger G, and Moseley ME. Gender differences in cerebral blood flow and oxygenation response during focal physiologic neural activity. J Cereb Blood Flow Metab 19: 1066-1071, 1999[Web of Science][Medline].

34.   Kearney, MT, Backofen JE, Koehler RC, Jones MD, and Traystman RC. Effect of hypoxemia on the cardiovascular response to intracranial hypertension in postnatal lambs. Am J Physiol Heart Circ Physiol 265: H1557-H1563, 1993[Abstract/Free Full Text].

35.   Komesaroff, PA, Black CVS, and Westerman RA. A novel, nongenomic action of estrogen on the cardiovascular system. J Clin Endocrinol Metab 83: 2313-3216, 1998[Abstract/Free Full Text].

36.   Kostrzewska, A, Laudanski T, and Batra S. Potent inhibition by tamoxifen of spontaneous and agonist-induced contractions of the human myometrium and intramyometrial arteries. Am J Obstet Gynecol 176: 381-386, 1977.

37.   Lipton, A. The anti-estrogen tamoxifen is a calcium antagonist in perfused rat mesentery. Cancer Chemother Pharmacol 20: 125-127, 1987[Web of Science][Medline].

38.   Magness, RR, Phernetton TM, and Zeng J. Systemic and uterine blood flow distribution during prolonged infusion of 17beta estradiol. Am J Physiol Heart Circ Physiol 275: H731-H743, 1998[Abstract/Free Full Text].

39.   Marynick, SP, Havens WW, Ebert MH, and Loriaux DL. Studies on the transfer of steroid hormones across the blood-cerebrospinal fluid barrier in the Rhesus monkey. Endocrinology 99: 400-405, 1979[Abstract/Free Full Text].

40.   McCall, AL, Han SJ, Millington WR, and Baum MJ. Non-saturable transport of [3H]oestradiol across the blood-brain barrier in female rats is reduced by neonatal serum. J Reprod Fertil 61: 103-108, 1981[Abstract/Free Full Text].

41.   McNeill, AM, Leslie FM, Krause DN, and Duckles SP. Gender difference in levels of alpha2-adrenoceptor mRNA in the rat tail artery. Eur J Pharmacol 366: 233-236, 1999[Web of Science][Medline].

42.   Mendelsohn, ME, and Karas RH. Estrogen and the blood vessel wall. Curr Opin Cardiol 9: 619-626, 1994[Web of Science][Medline].

43.   Mikkola, T, Ranta V, Orpana A, Ylikorkals O, and Viinikka L. Effect of physiological concentrations of estradiol on PGI2 and NO in endothelial cells. Maturitas 25: 141-147, 1996[Web of Science][Medline].

44.   Murakami, K, Nakagawa T, Shozu M, Uchide K, Koike K, and Inoue M. Changes with aging of steroidal levels in the cerebrospinal fluid of women. Maturitas 24: 71-80, 1999.

45.   Orstead, KM, Hess DL, and Spies HG. Pulsatile patterns of gonadotropins and ovarian steroids during estrus and pseudopregnancy in the rabbit. Biol Reprod 38: 733-743, 1988[Abstract].

46.   Palmon, SC, Williams MJ, Littleton-Kearney M, Traystman RJ, Kosk-Kosicka D, and Hurn P. D. Estrogen increases cGMP in selected brain regions and in cerebral microvessels. J Cereb Blood Flow Metab 18: 1248-1252, 1998[Web of Science][Medline].

47.   Pardridge, WM, and Mietus LJ. Regional blood-brain barrier transport of the steroid hormones. J Neurochem 33: 579-581, 1979[Web of Science][Medline].

48.   Pardridge, WW, Mietus LJ, Frumar AM, Davidson BJ, and Judd HL. Effects of human serum on transport of testosterone and estradiol into rat brain. Am J Physiol Endocrinol Metab 239: E103-E108, 1980[Abstract/Free Full Text].

49.   Pardridge, WW, and Mietus LJ. Transport of thyroid and steroid hormones through the blood-brain barrier of the newborn rabbit: primary role of protein-bound hormone. Endocrinology 107: 1705-1710, 1980[Abstract/Free Full Text].

50.   Pardridge, WW, and Mietus LJ. Transport of steroid hormones through the rat blood-brain barrier. Primary role of albumin-bound hormone. J Clin Invest 64: 145-154, 1979.

51.   Pelligrino, DA, Santizo R, Baughman VL, and Wang Q. Cerebral vasodilating capacity during forebrain ischemia: effects of chronic estrogen depletion and repletion and the role of neuronal nitric oxide synthase. Neuroreport 9: 3285-3291, 1998[Web of Science][Medline].

52.   Perrot-Applanat, M. Estrogen receptors in the cardiovascular system. Steroids 61: 212-215, 1996[Web of Science][Medline].

53.   Rodriguez, G, Warkentin S, Risberg J, and Rosadini G. Sex differences in regional cerebral blood flow. J Cereb Blood Flow Metab 8: 783-789, 1988[Web of Science][Medline].

54.   Shaul, PW. Rapid activation of endothelial nitric oxide synthase by estrogen. Steroids 64: 28-34, 1999[Web of Science][Medline].

55.   Shaw, TG, Mortel KF, Meyer J, Rogers RL, Hardenberg J, and Cutia MM. Cerebral blood flow changes in benign aging and cerebrovascular disease. Neurology 34: 855-862, 1984[Abstract/Free Full Text].

56.   Toscano, G, Sancesario G, Bianchi P, Cicardi C, Casilli D, and Giacomini P. Cerebrospinal fluid estrone in pseudotumor cerebri: a change in cerebral steroid hormone metabolism? J Endocrinol Invest 14: 81-86, 1999.

57.   Valverde, MA, Rojas P, Amigo J, Cosmelli D, Orio P, Bahamonde MI, Mann C, Vergara GE, and Latorre R. Acute activation of maxi-K channels (hSlo) by estradiol binding to the beta  subunit. Science 285: 1929-1931, 1999[Abstract/Free Full Text].

58.   Venkov, CD, Rankin AB, and Vaughn DE. Identification of authentic estrogen receptor in cultured endothelial cells: a potential mechanism for steroid hormone regulation of endothelial function. Circulation 94: 727-733, 1996[Abstract/Free Full Text].

59.   White, MM, Zamudio S, Stevens T, Tyler R, Lidenfeld J, Leslie K, and Moore L. Estrogen, progesterone and vascular reactivity: potential cellular mechanisms. Endocr Rev 16: 739-751, 1995[Abstract/Free Full Text].

60.   Wickoff, MH, Yuhanna IS, Pace MC, Mendelsohn ME, and Shaul PW. Plasma membrane-associated estrogen receptors mediate the acute activation of eNOS by estrogen. Circulation 98: 1312, 1998.

61.   Wu, CH, Blasco L, Flickinger G, and Mikhail G. Ovarian function in the preovulatory rabbit. Biol Reprod 17: 304-308, 1977[Abstract].

62.   Zancan, V, Santagati S, Bolego C, Vegato E, Maggi A, and Puglisi L. 17beta -estradiol decreases nitric oxide synthase II synthesis in vascular smooth muscle cells. Endocrinology 140: 2004-2009, 1999[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 279(3):H1208-H1214
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