(Stroke. 2000;31:738.)
© 2000 American Heart Association, Inc.
Original Contributions |
Deficient Mice
From the Departments of Anesthesiology and Critical Care Medicine (K.S., S.G., N.J.A., M.S., R.J.T., P.D.H.) and of Pathology (B.J.C.), Johns Hopkins University School of Medicine, Baltimore, Md; the Laboratory of Reproductive and Developmental Toxicology (K.S.K.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC; and the Department of Psychology (G.E.D., R.J.N.), Johns Hopkins University, Baltimore, Md.
Correspondence to Patricia D. Hurn, PhD, Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, 600 N Wolfe St, Blalock 1404, Baltimore MD 21287. E-mail phurn{at}jhmi.edu
| Abstract |
|---|
|
|
|---|
(ER
) is important to estrogens signaling in
the ischemic brain, then ER
-deficient (knockout) (ER
KO)
female mice would sustain exaggerated cerebral infarction damage after
middle cerebral artery occlusion.
MethodsThe histopathology of cresyl violetstained tissues was
evaluated after reversible middle cerebral artery occlusion (2 hours,
followed by 22 hours of reperfusion) in ER
KO transgenic and
wild-type (WT) mice (C57BL/6J background strain). End-ischemic
cerebral blood flow mapping was obtained from additional female murine
cohorts by using [14C]iodoantipyrine
autoradiography.
ResultsTotal hemispheric tissue damage was not altered by ER
deficiency in female mice: 51.9±10.6 mm3 in ER
KO
versus 60.5±5.0 mm3 in WT. Striatal infarction was
equivalent, 12.2±1.7 mm3 in ER
KO and
13.4±1.0 mm3 in WT mice, but cortical infarction was
paradoxically smaller relative to that of the WT (20.7±4.5
mm3 in ER
KO versus 30.6±4.1 mm3 in
WT). Intraocclusion blood flow to the parietal cortex was higher in
ER
KO than in WT mice, likely accounting for the reduced infarction
in this anatomic area. There were no differences in stroke outcomes by
region or genotype in male animals.
ConclusionsLoss of ER
does not enhance tissue damage in the
female animal, suggesting that estrogen inhibits brain injury by
mechanisms that do not depend on activation of this receptor subtype.
Key Words: estrogen cerebral ischemia gender menopause stroke
| Introduction |
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|
|
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As an initial step in understanding how estrogen signaling alters
cerebral ischemic injury, the contribution of the steroids
classic intracellular receptors has come under
investigation.18 19 20 Two subtypes of the estrogen receptor
(ER) are present and biologically active in the
brain21 22 and act as ligand-activated
transcription factors that alter gene expression in target cells: ER
and the recently identified ERß.23 24 Generalized
pharmacological ER blockade with pure antiestrogens exacerbates
ischemic injury in wild-type (WT) mice19 and
blocks estrogen-induced neuroprotection in cultured
neurons.20 However, studies with presently available
ER antagonists can be criticized on the grounds of the lack
of subtype specificity and poor bioavailability to the brain in vivo.
In the current study, we examined histopathological outcomes after
middle cerebral artery (MCA) occlusion and the regulation of cerebral
blood flow in ischemic and nonischemic brain in a
transgenic mouse strain deficient in ER
, known as ER knockouts
(ER
KO). As previously reported,25 26 the start codon
and amino-terminal domain of the gene are disrupted in these mice,
yielding a small expression of incomplete ER
transcripts but no
functional
-subtype receptors. ER
KO homozygotes of both sexes are
healthy but have abnormal reproductive function and sex
behavior.27 We demonstrate herein that loss of ER
does
not enhance perfusion defects after vascular occlusion or increase
tissue damage after ischemic stroke in female ER
KO mice,
suggesting that this ER subtype does not mediate estrogens
neuroprotective activity.
| Materials and Methods |
|---|
|
|
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KO mice and compared with that of WT controls (C57BL/6J
background strain; Harlan, Indianapolis, Ind) 1 week before MCA
occlusion. These tests assessed balance (time to fall from a narrow
pole up to a maximum of 120 seconds), agility (turning in a blind alley
or on an inclined screen), forelimb strength (hanging from suspended
wire), and autogrooming time.28 29 Cerebral ischemia was then induced by reversible MCA occlusion in these animals, as previously published.19 30 In brief, mice were anesthetized with 1% to 1.2% halothane in O2-enriched air by face mask, and rectal and temporalis muscle temperatures were controlled at 37±0.5°C throughout the experiment with heating lamps and water pads. Unilateral MCA occlusion was performed by inserting a 6-0 nylon monofilament into the internal carotid artery via an external carotid artery stump and then positioning the filament tip for occlusion at a distance of 6 mm beyond the internal carotid/pterygopalatine artery bifurcation. After securing the filament in place, the surgical site was sutured closed and infiltrated with 0.5% buvivacaine as needed for postoperative analgesia. The animal was then awakened and grossly assessed for neurological damage as follows: 0=no deficit, 1=failure to extend forelimb, 2=circling, 3=unilateral weakness, 4=no spontaneous motor activity. Mice with clear neurological deficits were reanesthetized with halothane for suture removal at 2 hours of occlusion. At 22 hours of reperfusion, each animal was again reanesthetized for transcardial perfusion with normal saline followed by neutral buffered 10% formalin. The brain was then postfixed in formalin and 30% sucrose in phosphate buffer, cut as serial coronal sections (40 µm) on a freezing microtome, and stained with cresyl violet. A set of 12 evenly spaced sections through the forebrain was mounted for determination of infarction volume by image analysis (Inquiry, Loats Inc). The following areas were measured in each section: cortical infarct, total ipsilateral cortex, total contralateral cortex, striatal infarct, total ipsilateral striatum, and total contralateral striatum. Because larger infarcts were associated with significant edema, areas in each section were corrected for edema as follows. The relative size of the cortical infarct was expressed as a percentage: 100%x[contralateral cortex-(total ipsilateral cortex-cortical infarct)]/ipsilateral cortex. The relative size of each striatal infarct was similarly corrected. Corresponding volumes were then calculated for the total set of slices. All measurements were carried out by an investigator blinded to treatment assignment.
Physiological measurements were carried out in
separate, age-matched ER
KO and WT animal cohorts. Femoral
arterial blood pressure and cortical laser-Doppler
flowmetry ([LDF] Moor Instruments Ltd) were determined during
occlusion and the first 30 minutes of reperfusion. A shallow
indentation was made in the parietal skull (2 mm posterior, 3
mm lateral to the bregma) with a low-speed drill for placement of the
LDF probe (DP3 optical, 1-mm diameter). A thin oil interface and the
probe were applied with a hood to block ambient light. The LDF signal
was recorded semicontinuously and averaged over 15-minute intervals
for comparison among treatment groups. Arterial blood
samples via femoral catheter (100-µL sample volume) were
analyzed for pH, PO2,
PCO2, and standard base excess at
baseline and at end-ischemia.
In an additional set of female animals, regional cerebral blood flow was measured by [14C]iodoantipyrine autoradiography, as previously described5 14 and modified for the mouse. Mice with clear neurological deficits during MCA occlusion were reanesthetized, and arterial (Clay Adams PE 10; 0.28-mm ID, 0.61-mm OD, 15 cm long) and venous (PE 10; 10 cm long) femoral catheters were inserted. At 120 minutes of MCA occlusion, arterial blood pressure, pH, PCO2, and PO2 were measured, and intravenous infusion and arterial sampling were started. A total of 4 µCi of [14C]iodoantipyrine in 81 µL of isotonic saline was infused intravenously over 45 seconds at a constant infusion rate of 6.48 mL/h. Simultaneously, the arterial catheter was opened, and blood was allowed to flow freely into heparinized saline drops of known volume placed in paraffin wells. Nine blood samples were collected at 5-second intervals; the mouse was decapitated at 45 seconds; and the brain was quickly removed (<60 seconds), frozen in 2-methylbutane on dry ice, and stored at -80°C. Each brain was sectioned on a cryostat (20-µm-thick coronal sections at -18°C) and thaw-mounted onto glass coverslips. Sections were apposed for 1 week to film (Kodak, SB-5) with 14C standards. Sample volume was measured by using a pipette and calculated by subtracting the volume of a saline drop from the total volume of blood sample plus saline. Parallel time-control saline drops were used to account for changes in volume due to evaporation. The concentration of [14C]iodoantipyrine was determined by liquid scintillation spectroscopy after decolorization with 0.2 mL of tissue solubilizer (Soluene-350, Packard Instruments Co). Autoradiographic images representing 7 coronal levels (+4, +3, +2, +1, 0, -1, and -2 mm from the bregma; 3 images each) were digitized, and regional cerebral blood flow was determined by image analysis software (Inquiry, Loats Associates). Rates of regional cerebral blood flow were calculated at discrete 0.1-mm2 regions within cortical and subcortical regions within the MCA distribution and averaged over 3 to 7 consecutive coronal slices; the images of each coronal slice were scanned and pixels were stratified according to corresponding blood flow rates. Pixels with flow rates falling within a range of blood flow were summed and converted to volume units.14
All data are expressed as mean±SEM. Statistical evaluation was
performed by Students t test to compare infarction volumes
and regional cerebral blood flow between animal groups.
Physiological and behavioral variables were
analyzed by 2-way ANOVA and a post hoc Newman-Keuls test to
determine differences between groups. Postischemic
neurological scores were analyzed by the Mann-Whitney
U test. The criterion for statistical significance was set
at P
0.05.
| Results |
|---|
|
|
|---|
KO animals of both sexes demonstrated no
abnormalities. There were no differences among groups in gross
neurological score as assessed during MCA occlusion (2.4±0.2 and
2.6±0.2 in WT and ER
KO female mice; 2.1±0.2 and 2.2±0.2 in WT and
ER
KO male mice). Total tissue damage within the
ischemic hemisphere was unchanged by ER
deficiency in
females: hemispheric infarction volume was 51.9±10.6
mm3 in ER
KO females versus 60.5±5.0
mm3 in WT females. Similarly, striatal injury was
equivalent: 12.2±1.7 mm3 in ER
KO and
13.4±1.0 mm3 in WT mice. Only cortical
infarction was altered in ER
KO females: it appeared paradoxically
less (20.7±4.5 mm3) than would be
anticipated from corresponding measurements in WT mice (30.6±4.1
mm3). Figure 1
deficiency (1.6±0.2 in ER
KO versus 1.9±0.1 in WT). There were no
differences in stroke outcomes by region or genotype in male
ER
KO versus WT animals. Arterial blood pressure and
respiratory gas composition were monitored before and during MCA
occlusion and were comparable among groups (the
Table
|
|
To map cortical and subcortical perfusion deficits,
intraischemic blood flow ipsilateral and contralateral to the
occlusion was quantified throughout the brain at 2 hours of MCA
occlusion (Figure 2
). The distribution of
tissue volume recruited into near-zero and low-flow zones within the
ischemic hemisphere was not different in ER
KO and WT females
(Figure 3
), suggesting a similarity of
ischemic insult. Absolute blood flow in all regions evaluated
within the nonischemic hemisphere was equivalent in ER
KO and
WT mice, indicating that the loss of ER
does not alter baseline
cerebral blood flow in the female. Furthermore, intraischemic
blood flow was not different between groups in all brain regions
examined, with the exception of the parietal cortex (Figure 4
). Flow to this area during occlusion
was elevated in ER
KO relative to WT females, likely accounting for
our observation of reduced infarction in this anatomic area. In
addition, LDF data obtained over the parietal cortex suggested that
localized perfusion was less severely reduced throughout occlusion in
ER
KO females (Figure 5
).
|
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| Discussion |
|---|
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|
|---|
was important to estrogens
signaling in the ischemic brain, then ER
KO mice would
sustain an exaggerated cerebral infarction after MCA occlusion. The
main finding of the study is that loss of ER
neither enhances tissue
damage in the female animal nor exacerbates intraischemic
tissue perfusion defects. Total hemispheric infarction was unchanged in
ER
KO relative to age-matched WT mice of the same background strain.
These data suggest that estrogen inhibits brain injury by mechanisms
that do not depend on activation of the ER
subtype. Alternative
signaling pathways include activation of the intracellular ERß
subtype or nonreceptor-initiated mechanisms.
Clinical ischemic stroke is frequently the sequela of
atherothrombotic vascular occlusion, with varying degrees of persistent
tissue perfusion from collateral and anastomotic microvessels.
Endogenous brain protectants may therefore act by 1 or both
of 2 distinct pathophysiological mechanisms: by
maximally dilating collateral circulation and partially ameliorating
intraocclusion loss of blood flow or by direct cell preservation of
parenchymal neurons and glia. Previous work5 8 11
emphasized that endogenous estrogen utilizes both
approaches to salvage brain tissue in the female after experimental
ischemic stroke. We used a novel transgenic strain to dissect
the role of 1 ER subtype in cerebrovascular pathophysiology. Currently
available pharmacological antiestrogens do not provide receptor
subtypeselective antagonism; therefore, ER
KO animals have provided
many new insights into estrogens signaling mechanisms in a variety of
tissue and cell types (for a review, see Reference 2727 ). The present
results suggest that ER
-mediated mechanisms are not important to
tissue outcome in experimental stroke.
Estrogens clearly have direct and rapid effects on nonreproductive
neuronal tissue and on the cerebral vasculature. For example, synaptic
architecture within areas such as the hippocampus changes with the
estrous cycle and can be altered in <24 hours by exogenous
estradiol.31 Furthermore, complementary fluctuations in
the volume of astrocytic processes and synaptic numbers occur in
response to ovarian steroids.32 The steroid may
utilize diverse signaling pathways to produce biological effects. These
include (1) nuclear ER-linked modulation of target gene transcription
efficiency; (2) ER-dependent but nontranscriptional mechanisms; (3)
nonER-linked transcriptional mechanisms that utilize generalized
signaling molecules; and (4) cell membraneassociated activity that is
far too rapid to involve mRNA transcription and protein synthesis (for
recent reviews, see References 33 and 3433 34 ).In addition, cross-talk
between membrane-mediated events and nuclear receptor activation has
also been described, particularly within the
vasculature.34 There are few data that distinguish which
of these signaling modalities are used by estrogen to initiate (or
integrate) its many putative anti-ischemic mechanisms. Such
mechanisms include induction of neuroprotective gene products
bcl218 35 36 and neurotrophic growth
factors,37 38 nontranscriptional modulation of excitatory
neurotransmission and glutamate toxicity,20 39 40 and
antioxidant activity.41 42 The present data allow the
exclusion of 1 signaling pathway by which estrogen acts in
ischemic brain: nuclear ER
activation. Although deficiency
in this subtype does not exacerbate histological damage
in females, we have recently observed increased damage after MCA
occlusion in WT female mice chronically treated with ICI 182,780, an
inhibitor of both known ER subtypes.19
Therefore, it is likely that loss of functional ERß, rather than
ER
, is responsible for amplifying stroke damage in these mice.
We and others have also observed that endogenous estrogen
amplifies residual cerebral blood flow in female animals during
vascular insult or occlusion.5 8 11 Such promotion of
blood flow during ischemic stress is lost in estrogen-deficient
animals and is absent in the male.5 8 11 Potential
mechanisms include estrogen-induced increases in vascular diameter;
enhanced vasodilatory capacity through increased elaboration of nitric
oxide, prostacyclin, or other endothelium-derived
mediators; and reduced sensitivity to selected vasoconstrictor stimuli.
Such observations are not surprising, since estrogen has well-known
vasoactive properties in the cerebral circulation. Gray-matter blood
flow is higher in women versus men,43 44 45 but sex
differences disappear by 50 to 60 years of age.43 44
Premenopausal women demonstrate greater cerebral vasodilatory capacity
to stimuli such as increased systemic hypercapnia when compared with
men of the same age.46 Exogenous estrogen replacement also
increases blood flow throughout brain regions, including the cortex,
cerebellum, basal ganglia, and hippocampus and produces cerebral
vasodilation in animals47 48 and humans
with49 and without50 significant
cerebrovascular disease. However, the current findings indicate that
cerebral blood flow is not depressed in the healthy brain or within the
ischemic lesion in ER
KO females relative to WT C57BL/6J
mice, suggesting that estrogens basal or stress-evoked vasodilator
properties are not likely dependent on ER
.
An unanticipated finding was the selective reduction of cortical injury
observed in ER
KO females, potentially explained by a relative
preservation of intraocclusion blood flow to the parietal cortex.
Whether this result represents a unique response to loss of the
receptor subtype is unclear; however, the anatomic limitation (parietal
cortex only) and sex bias (females only) would argue against a
nonspecific compensatory physiology within the transgenic strain. A
plausible explanation for this finding is related to the chronically
elevated plasma estrogen levels sustained in the ER
KO female,
consistent with their hormone insensitivity (84 pg/mL,
3
times that of the WT female mouse).27 Because estradiol is
vasoactive, high endogenous levels may have improved
outcome by flow-dependent, ER
-independent means in steroid-sensitive
cortical regions. We have previously observed intraocclusion
preservation of cerebral blood flow in WT female rodents and
rabbits.5 8 If so, such protective effects could be
mediated by the recently identified ERß and/or by nonreceptor,
membrane-associated binding to target cells. Although expression of
ERß in cerebral vessels has not yet been shown, ERß mRNA is
present in the ER
KO aorta51 and is induced by
vascular injury in both endothelial and vascular smooth
muscle cells.52 Furthermore, ERß mRNA is present in
the cortex of ER
KO mice,21 and there is evidence of
translation into a 17ß-estradiolbinding, biologically active
protein.22
In conclusion, ER
deficiency does not enhance tissue damage in
female animals, indicating that estrogen inhibits brain injury by
mechanisms that do not depend on activation of this receptor subtype.
Our findings may have clinical relevance to the current search for
selective estrogen receptor agonists that are useful hormone
replacement agents from the perspective of bone and heart but that have
adjunctive neuroprotective properties. Such agents could be helpful to
women who elect estrogen therapy in their middle years but who also
carry the risk for or a history of ischemic stroke and
cerebrovascular disease. This animal study would argue against
targeting ER agonists with selective ER
activity in the brain.
| Acknowledgments |
|---|
Received October 26, 1999; revision received December 6, 1999; accepted December 27, 1999.
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Lindner V, Kim SK, Karas RH, Kuiper GGJM, Gustafsson
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receptor-ß mRNA in male blood vessels after vascular injury.
Circ Res. 1998;83:224229.
Department of Pharmacology, College of Medicine, University of California, Irvine
| Introduction |
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|
|
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and ß, expands possibilities for development of more selective
therapies. Transgenic mice deficient for the
- or ß-estrogen
receptor have already been used to demonstrate differential actions of
estrogen mediated by each receptor type.
Protective effects of estrogen have been well documented in
animal models of stroke. The present study by Sampei et al clearly
demonstrates that these actions of estrogen are not solely dependent on
the
-estrogen receptor. Whether protective effects of estrogen are
mediated by the ß-receptor or by another as yet undescribed mechanism
remains to be determined. Further delineation of the nature of the
estrogen receptor involved will contribute to better understanding of
the mechanism of estrogens protective effect and, perhaps, improved
prevention and/or treatment of stroke.
Received October 26, 1999; revision received December 6, 1999; accepted December 27, 1999.
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