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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
TOP
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
TOP
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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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