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From the Department of Anesthesiology and Critical Care Medicine, Johns
Hopkins University School of Medicine (N.J.A., I.H., R.J.T., P.D.H.) and
National Institute on Drug Abuse (A.S.K., E.D.L.), Baltimore, Md.
Correspondence to Patricia D. Hurn, PhD, Department of Anesthesiology and Critical Care Medicine, Johns Hopkins Medical Institutions, 600 N Wolfe St/Blalock 1404, Baltimore, MD 21287. E-mail phurn{at}welchlink.welch.jhu.edu
Methods—Age-matched male (M), intact female (F), and
ovariectomized female (O; plasma estradiol: 4.1±1.6 pg/mL compared
with 7.4±1.5 in F and 4.0±1.1 in M) rats from two different strains,
normotensive Wistar and stroke-prone spontaneously hypertensive rats,
were subjected to 2 hours of intraluminal middle cerebral artery
occlusion, followed by 22 hours of reperfusion. Cerebral blood flow
(CBF) was monitored throughout the ischemic period by
laser-Doppler flowmetry. Infarction volume in the cerebral
cortex (Ctx) and caudoputamen (CP) was determined by
2,3,5-triphenyltetrazolium chloride
staining. In a separate cohort of M, F, and O Wistar rats, absolute
rates of regional CBF were measured at the end of the ischemic
period by quantitative autoradiography using
[14C]iodoantipyrine.
Results—F rats of either strain had a smaller infarct size in Ctx
and CP and a higher laser-Doppler flow during ischemia
compared with respective M and O rats. Mean end-ischemic CBF
was higher in F compared with M and O rats in CP, but not in Ctx.
Cerebrocortical tissue volume with end-ischemic CBF <10 mL/100
g/min was smaller in F than M rats, but not different from O rats.
Conclusions—We conclude that endogenous estrogen
improves stroke outcome during vascular occlusion by exerting both
neuroprotective and flow-preserving effects.
A growing body of evidence indicates that estrogen has multiple
vascular effects,10 11 all of which could contribute to
salvage of neural tissue during ischemic episodes. Single
reports indicate that estrogen increases cerebral perfusion in women
with12 or without13 known cerebrovascular
disease. Furthermore, we have previously shown that residual cerebral
blood flow during global cerebral ischemia in animals can be
aug- mented by chronic estradiol treatment.14 It has been
proposed that estrogen has tissue antioxidant properties, which could
also contribute to neuroprotection during ischemic
episodes.5 We now utilize a stroke model of transient focal
ischemia in two genetically distinct strains of rat to compare
ischemic outcome between males and females and examine the role
of endogenous female sex hormones in any gender-specific
responses. We also measured regional cerebral blood flow during
vascular occlusion to determine the contribution of flow preservation
in gender-specific stroke outcome.
Ovariectomy was performed at the age of 10 to 12 weeks. Briefly, under
halothane anesthesia (1% to 2% via snout mask in
O2-enriched air), the ovary was accessed through a lateral
abdominal incision, and the ovarian artery and vein were
clamped with a fine surgical hemostat and ligated. The ovary was then
resected, surgical wounds closed, and the animal allowed to recover for
2 to 4 weeks. On the day of the experiment, rats were
anesthetized as above and instrumented with a femoral artery
catheter for monitoring arterial blood pressure and
measurement of blood gases. Rectal and temporalis muscle temperatures
were controlled at 37±0.5°C using heating lamps. An area 2 to 3
mm in diameter was thinned in the right parietal bone 2-mm posterior
and 6-mm lateral to the bregma for placement of a LDF probe (Moor
Instruments, Ltd., model MBF3D, England), as previously
described.17 To allow for continuous monitoring of LD-CBF,
the head piece of a stereotaxic frame was modified to allow
for free rotation around the longitudinal axis of the rat and was
equipped with a snout mask for ventilation and with a holder for the
LDF probe. The probe was positioned during the control period over an
area devoid of visible blood vessels, and its position was not changed
throughout the experiment. The signal was allowed to stabilize over a
30-minute period before a baseline reading was taken before vascular
occlusion.
MCA occlusion was achieved by modifying an established procedure for
proximal occlusion of MCA in the rat with an intraluminal
filament.18 19 20 Briefly, the right common carotid artery
was exposed through a lateral incision, carefully separated from the
vagus and ligated. The external carotid artery was ligated, the
occipital branch cauterized, and the pterygopalatine artery exposed and
ligated. An occluding filament (4–0 monofilament nylon surgical suture
with a heat-rounded tip) was advanced into the internal carotid artery
until the LDF signal displayed an abrupt and significant reduction
(77%±2% from baseline, n=62); the filament was then secured in
place. Measurements were made over 5 minute periods at 5, 15, 30, 60,
90, and 120 minutes after occlusion. Withdrawal of the string at the
end of the ischemic period (2 hours) was associated with a
rapid restoration of flow. LD-CBF reached 66%±7%, 75%±10%, and
90%±6% of baseline by 5 minutes, and 79%±8%, 93%±7%, and
87%±11% of baseline by 15 minutes after withdrawal of the filament
in M (n=20), F (n=22), and O (n=20), respectively. After 22 hours of
reperfusion, the animal was reanesthetized with 3% halothane,
and a blood sample withdrawn for determination of plasma hormone
levels. Plasma 17-ß-estradiol and progesterone were measured in
triplicate by radioimmunoassay, as previously described.14
The brain was harvested and sliced into seven 2-mm thick coronal
sections for TTC staining, as previously described.18
Infarction volumes were measured using digital photography and image
analysis software (SigmaScan Pro, Jandel). The infarcted area
(unstained) was numerically integrated across each section and over the
entire ipsilateral hemisphere. Infarct volume was measured separately
in the cerebral cortex and caudoputamen and expressed as a
percentage of the volume of the ipsilateral side.
End-ischemic rCBF was measured in additional cohorts of Wistar
rats using quantitative autoradiography with
[14C]IAP, as described previously.21 22
Animals were instrumented with femoral vascular catheters, and the MCA
occluded as in the previous cohorts. At 2 hours of MCA occlusion,
arterial blood pressure and blood gases were measured, then
40 µCi of [14C]IAP (New England Nuclear) in 1 mL of
isotonic saline were infused intravenously for 45 seconds.
During infusion, fifteen 20-µl samples of free-flowing
arterial blood from the femoral artery catheter were
collected in heparin-coated sample tubes. With the filament still in
place and the laser-Doppler indicating ischemic status, the
rat was decapitated 45 seconds after the start of infusion. The brain
was quickly removed and frozen at -50°C in 2-methylbutane on dry
ice. Each brain was cryostatically sectioned into 20-µm-thick coronal
sections at -20° and thaw-mounted onto cover glasses. Sections were
apposed for 1 week to film (Kodak, SB-5) with 14 C
standards. The concentration of [14C]IAP in blood samples
was determined by liquid scintillation spectroscopy (Beckman, model
3801) after decolorization with 0.2 mL of tissue solubilizer
(Soluene-350, Packard Instruments Co., Downers Grove, Ill).
Autoradiographic images representing three
different coronal levels (+2.7, +0.2, and -1.8 mm from the
bregma,23 six to nine images each) were digitized and
regional CBF determined using image analysis software (Inquiry,
Loats Associates, Westminster, Md). Rates of rCBF were calculated by
the Kety-Schmidt modification of the Fick principle:
Two methods of analysis were used to determine rCBF. First, CBF
was measured by sampling 0.08-mm2 squares within those
regions most vulnerable to MCA occlusion, the frontal and parietal
lobes of the cerebral cortex and the medial and lateral aspects of the
caudoputamen complex. Flow rates were then averaged from
squares assayed from six to nine consecutive brain slices at each of
three coronal levels. In the second method, areas with flow rates below
10 mL/100 g/min were isolated by digital image scanning and
perimetrically measured for each slice in the entire ischemic
hemisphere and in the cerebral cortex alone. Areas were averaged over
six to nine images from each brain level and then were numerically
integrated across the three coronal levels to obtain an estimate of
tissue volume with severely compromised ischemic blood
flow.
All values are reported as means±standard errors of the mean unless
otherwise indicated. Physiological
parameters were subjected to two-way ANOVA. Differences in
infarct size, mean residual laser-Doppler flow and
autoradiographic cerebral blood flow among groups were
determined with one-way ANOVA. Post hoc comparisons were made with
Newman-Keuls test. The relationship between residual LD-CBF and infarct
size was examined by regression analysis. The criterion for
statistical significance is P
Fig 1
The incidence of stroke is lower in premenopausal women relative to men
of the same age.1 2 3 However, little is known about stroke
outcome and ischemic mechanisms in males versus females once
stroke has taken place and tissue injury is in process. We used an
established animal model of reversible occlusion of middle cerebral
artery to compare outcome in male and female rats from two genetically
distinct strains and to explore the underlying mechanism of any
gender-linked ischemic brain injury. Our data indicate that
estrogen-primed female rats sustain strikingly smaller brain infarcts,
both in the cerebral cortex and striatum, than age-matched males. This
observation was consistent across strains and valid even in
animals with baseline hypertension, a known risk factor for stroke and
vascular pathology. Sex-linked differences in stroke outcome in SHR-SP
strain are of particular importance given the similarities between
underlying pathology of this strain and human stroke.15
When corresponding groups from the two strains of rats are compared,
SHR-SP rats suffer more severe brain damage compared with Wistar rats.
This finding agrees with previous reports that vascular occlusion in
genetically hypertensive strains of rat results in larger infarct
volume than in normotensive strains.24 This observation
suggests that salvage of neural tissue associated with estrogen is
likely to be a generalizable phenomenon despite quantitative
differences in the magnitude of tissue damage among individual
strains.
Our findings with MCA occlusion in the rat are consistent with
previous work demonstrating gender-specific responses to experimental
ischemic brain injury.5 6 Female gerbils are less
vulnerable to hippocampal neuronal injury after unilateral carotid
occlusion than their male counterparts, consuming smaller amounts of
endogenous antioxidants during the ischemic
insult.5 Li et al have demonstrated differences in severe
cortical infarcts, defined as the amount of cytopathology and loss of
astrocytic staining, between male and female Fisher rats after
thromboembolic injury.6 Our findings expand on these
earlier observations by demonstrating the deleterious consequences of
endogenous estrogen deficiency in stroke and by evaluating
the contribution of flow-mediated and flow-independent mechanisms in
the more favorable outcome (sparing of neural tissue) enjoyed by female
animals after experimental stroke.
The gender difference in infarct size is lost when female animals are
ovariectomized at an early age. Ovariectomized female rats sustain a
similar infarct size as males, which indicates that salvage of neural
tissue in females is due to an action of female sex steroids, most
likely estrogen because ovariectomized females, which have plasma
estrogen levels similar to males loose the advantage enjoyed by intact
females and display infarct size similar to that of males. It is
unlikely that progesterone was responsible for the improved outcome of
stroke in intact versus ovariectomized females because plasma
progesterone in ovariectomized rats was lower than that in intact
females,but still higher than in males. However, in view
of recent reports demonstrating a neuroprotective effect of exogenously
administered progesterone in male rats,25 the possibility
of an interaction between the two hormones cannot be excluded. In
support of such an interaction is the demonstration of induction of
progesterone receptors by estrogen in the brain.26
Differences in stroke outcome could not be accounted for by differences
in age, halothane concentration, core or head temperatures, or
physiological variables such as
arterial blood pressure or blood gases, because these
parameters were similar among all groups within each
strain.
Ischemic status, as well as reperfusion, was confirmed in each
animal by continuous on-line monitoring of laser-Doppler (LD)-CBF.
LD-CBF during ischemia, an indication of residual tissue
perfusion after arterial occlusion, correlated well with
infarct size and was higher in F compared with M and O rats in both
strains. This observation suggests that differences in residual tissue
perfusion may account for gender differences in infarct size and that
estrogen acts to enhance tissue perfusion during vascular occlusion.
The latter view agrees with our previous finding that chronic exogenous
administration of 17-ß-estradiol, the principal biologically active
estrogen in mammals, augments residual CBF during global incomplete
ischemia in the rabbit.14 However, in a gerbil
model of global cerebral ischemia, differences in cortical CBF
were not apparent between males and females despite differences in
neuronal injury.5 Because LD-CBF estimates only the
relative change in CBF, we used 14C
autoradiography to quantify end-ischemic CBF
within MCA-dependent territory and to determine whether residual
regional CBF was indeed higher in intact females compared with males
and ovariectomized females. In caudoputamen,
end-ischemic CBF was clearly preserved in females when
endogenous estrogen is present compared with males or
ovariectomized females. Blood flow preservation is, therefore, a likely
explanation for the smaller infarction volume observed within this
region in estrogen primed females. It is unlikely that preservation of
CBF in the striatum is a reflection of differences in baseline CBF,
because flow in the nonischemic, contralateral region was
similar among the three groups. This conclusion is also
consistent with our previous observation that baseline blood
flow does not increase with exogenous estrogen
administration,14 at least not in anesthetized
animals. In conscious animals, acute estrogen administration has been
reported to increase CBF in many brain areas, including the cerebral
cortex, hippocampus, basal ganglia and cerebellum, both in males and
females.27 Using positron-emission tomography scanning,
estrogen has recently been shown to augment cognitive activation of
regional CBF in young women.28
In contrast to our finding in the caudoputamen,
end-ischemic cortical flow was not different among the three
groups, suggesting that flow-preservation is an unlikely explanation
for the differences observed in infarct size in this region. To further
dissect flow-mediated mechanisms in cortical tissue sparing in
estrogen-primed animals, we compared tissue volumes with rCBF of less
than 10 mL/100 g/min among groups, as a means of equalizing the
contribution of flow deficit to the development of cerebrocortical
tissue infarction. These regions with a blood flow rate approaching the
ischemic threshold for neuronal cell death29 30
represent areas of the cortex destined to infarction. Our
analysis indicates that despite a clear
histological preservation of viable tissue within
cortex in intact females versus males and ovariectomized females, the
volume of ischemic tissue was smaller in the cortex of intact
females compared with males, but the volume of similarly profoundly
ischemic tissue in the cortex of ovariectomized females was not
different from that of intact females. This finding suggests that flow
preservation is a likely explanation for the difference in infarct size
between males and females. However, the difference between intact and
ovariectomized females cannot be fully explained by flow-mediated
mechanisms, and likely represents a flow-independent
neuroprotective effect of estrogen. This process is apparent in that,
whereas estrogen-competent females have smaller areas with severely
ischemic flow than males, ovariectomy does not alter this
volume of severely low flow tissue destined for infarction. Thus, for
similar areas of severely ischemic flow, intact females sustain
smaller infarction volumes than do estrogen-depleted females.
Therefore, we conclude that non-flow–mediated mechanisms of
neuroprotection are at work in ischemic cortex in females.
Our finding that female animals maintain higher tissue perfusion during
MCA occlusion may be explained by one or more of the following
mechanisms: (1) cerebrovascular anatomical differences, eg, greater
number and/or diameter of cerebral blood vessels providing extensive
collateral flow in females; (2) increased small vessel and capillary
density in response to estrogen's angiogenic properties;31
(3) functional differences in vascular reactivity; or (4) availability
of vasoactive mediators, allowing for increased vasodilator capacity
under low flow conditions in estrogen-primed vessels. The latter two
mechanisms seem likely in that direct effects of estrogen on both
endothelium and vascular smooth muscle have been
reported.10 11 32
A direct effect of estrogen on the blood vessel wall is suggested by
its ability to relax isolated blood vessels10 11 and by
evidence for the presence of ER in isolated arteries and vascular
cells.33 Expression of classic ER has been demonstrated in
cultured human and bovine endothelial
cells,34 and estrogen-binding sites have been described in
human endothelial and rat aortic vascular smooth muscle
cells.35 Potential mechanisms for estrogen-induced
vasodilation include estrogen-mediated release of nitric oxide and
prostacyclin from vascular endothelium10
and VSM membrane hyperpolarization,36
possibly by cGMP-dependent phosphorylation of
K+ channels.37 Of interest in this regard is
the presence of estrogen-responsive elements on genes regulating
rate-limiting enzymes in the biosynthesis of both prostacyclin and
nitric oxide.10 Furthermore, vascular reactivity has been
shown to be impaired in ovariectomized animals, and it is restored by
chronic estrogen treatment.38 Thus, estrogen may protect
the brain during ischemic episodes by preserving and augmenting
vasodilator mechanisms.
Finally, the cortical neuroprotection observed in intact versus
ovariectomized females is likely to represent alternative and
significant nonvascular mechanisms. Estrogen exhibits an array of
neuronal effects that could potentially alleviate ischemic
brain damage, and there is a widespread pattern of ER and associated
estradiol targets within neural tissue.26 39 40 ER mRNA has
been identified in brain areas not generally associated with
reproductive function.41 Furthermore, glial cells
contain receptors for estrogen and respond to the hormone both in vivo
and in vitro.42
In summary, we have demonstrated that female rats sustain more
favorable outcome after ischemic brain injury, possibly due to
both neuroprotective and flow-preserving effects of estrogen. This
finding provides a possible explanation for the well-documented, but
poorly understood, clinical observation that premenopausal women are at
lower risk for stroke than men of the same age. Our finding may also
explain the steep rise in stroke incidence in women after menopause. It
is yet to be determined whether exogenously administered estrogen
interacts with mechanisms of ischemic brain damage to protect
neurons. Such a finding would provide a rationale for the use of
estrogen to improve stroke outcome in postmenopausal women.
Furthermore, understanding the mechanisms of estrogen-mediated
neuroprotection helps elucidate the generalized mechanisms of stroke
damage and provides new strategies for stroke injury prevention in both
sexes.
Received June 12, 1997;
revision received October 2, 1997;
accepted October 6, 1997.
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Director,
National Institute of Nursing Research,
National Institutes of Health,
Bethesda, Maryland
Although stroke has long been regarded as a problem of concern
predominantly in men, there is a steep increase in the incidence of
stroke in postmenopausal women. Moreover, since women have a longer
life expectancy than men, the incidence of stroke is higher in older
women. The mean age of our population is increasing. As we enter the
21st century a dramatic shift is occurring. As the population ages and
women comprise a larger percentage of the population, the issue of
gender difference in stroke will become even more important. Therefore,
it becomes imperative that we are able to address the problem of stroke
in both genders of our population.
Moreover, now that there is a newly available treatment for stroke,
issues such as the role that gender plays in the size of the lesion and
any changes in response to treatment must be characterized. Certain sex
hormones such as estrogen are known to have effects on the brain.
Effects such as alteration of regional cerebral blood flow, vasodilator
effects, augmentation of cognitive activation and increased neuronal
branching in vitro, and cognitive protection in Alzheimer's
disease have all been attributed to estrogen. All of this information
may play an important role in either augmenting or modifying response
to therapies instituted in the acute stroke situation in women.
Therefore, it is important to raise the question of and provide the
answers to the role, either beneficial or potentially deleterious, of
sex hormones that may influence the response to cerebral
ischemia.
This study addresses gender and its relation to brain injury in an
animal model of focal cerebral ischemia. The authors'
conclusions that endogenous estrogen improves stroke
outcome by providing both neuroprotective effects and blood flow
following focal cerebral ischemia are carefully discussed in
detail in the paper. The resulting information provides guidance as the
field of clinical stroke research moves forward aggressively in testing
therapeutic interventions to prevent and/or limit disabilities
resulting from stroke.
Received June 12, 1997;
revision received October 2, 1997;
accepted October 6, 1997.
© 1998 American Heart Association, Inc.
Original Contributions
Gender-Linked Brain Injury in Experimental Stroke
Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
Background and Purpose—Premenopausal
women are at lower risk than men for stroke, but the comparative
vulnerability to tissue injury once a cerebrovascular incident occurs
is unknown. We hypothesized that female rats sustain less brain damage
than males during experimental focal ischemia and that the
gender difference in ischemic outcome can be eliminated by
ovariectomy.
Key Words: cerebral blood flow • estrogens • stroke, experimental • gender • neuroprotection
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
The risk of stroke is
lower in premenopausal women relative to men of the same
age,1 2 but the incidence of cerebrovascular events rapidly
increases in women after menopause.3 Historically, these
epidemiological findings have been attributed to estrogen, in part due
to the beneficial effect of the hormone in preventing coronary
heart disease.4 However, the comparative vulnerability of
females and males to tissue injury once stroke is ongoing remains
understudied. There is some evidence for gender-specific responses to
experimental ischemia. Female gerbils have lower incidence of
and less severe brain lesions after carotid occlusion compared with
males.5 Furthermore, thromboembolism induced by
photochemical irradiation of the carotid artery differentially affects
male and female rats, producing greater inflammatory responses, but
less severe infarcts, in female than in male rats.6 There
is also evidence for gender-specific responses to other types of brain
injury, such as cerebral contusion,7
hypoxia,8 and drug-induced
toxicity.9
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
This study was conducted in accordance with the National
Institutes of Health guidelines for the care and use of animals in
research and the protocols were approved by the Animal Care and Use
Committee of the Johns Hopkins University. Age-matched, adult (13 to 15
weeks) male (M), intact female (F), and ovariectomized female (O) rats
from two strains, normotensive Wistar (255 to 360 g of body
weight, n=45) and stroke-prone spontaneously hypertensive rats (SHR-SP,
182 to 282 g, n=32), were studied. SHR-SP rats were included in
the study in view of the strain's genetic predisposition to stroke,
presence of hypertension, and overall similarities to human
stroke.15 A colony of SHR-SP is maintained in our
institution from a stock obtained from the National Institutes of
Health (Laboratory Sciences Section, Veterinary Resources Program,
National Center for Research Resources, Bethesda,
Md).16
where Cbrain is the concentration of the tracer in
the tissue at the time of decapitation (T),
Cblood is the concentration of the tracer in
arterial blood, t is the variable time and
K (the transfer coefficient)=(m) ·
(rCBF)/
, where rCBF is the rate of blood flow per unit mass of
tissue (mL/100 g/min), m is the diffusion equilibrium
constant (mIAP=1.0), and is the tissue:blood
partition coefficient (IAP=0.992). 
.05.
Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
Physiological data are summarized in Table 1
. The MAP was equivalent in all groups
within the same strain and was maintained at values similar to baseline
throughout the experimental protocol. Baseline MAP was higher in SHR-SP
rats compared with all corresponding groups from the Wistar strain.
Baseline arterial pH, PaCO2, and
PaO2, and hemoglobin and glucose concentrations
were equivalent among all groups and were maintained at values similar
to baselines throughout the experimental protocols. Plasma estradiol
concentrations were 7.36±1.11, 3.95±1.46, and 4.11±1.58 pg/mL, and
plasma progesterone concentrations were 27±2.8, 5.9±0.8, and 16±3.3
ng/mL in F (n=28), M (n=21), and O groups (n=17), respectively.
View this table:
[in a new window]
Table 1. Summary of Selected Physiological
Variables Before (B), During (D), and After (A) Middle Cerebral
Artery Occlusion summarizes infarction volumes as a
percentage of the cerebral cortex or caudoputamen in Wistar
rats. Male rats sustained larger infarcts than females both in the
cerebral cortex (25.7%±6.8% in M compared with 9.4%±2.9% in F)
and caudoputamen (41.5%±4.4% in M versus 19.9% ±4.8%
in F). Ovariectomized rats sustained injury volumes that were not
different from that of males (30.2%±6.4% in the cerebral cortex and
42.4%±4.0% in CP). Fig 2
represents averaged residual laser-Doppler CBF over the 2
hours of MCA occlusion in the same animals. Female rats maintained a
higher percentage of baseline LD-CBF during ischemia compared
with male and ovariectomized rats (37.6%±3.1% in F rats compared
with 29.7%±0.7% and 27.0%±1.2% in M and O, respectively). Similar
differences in infarct size and ischemic flow between genders
that are abolished by ovariectomy were also observed in SHR-SP rats.
Although SHR-SP rats had a significantly greater infarct size and a
more severe reduction in LD-CBF during ischemia compared with
corresponding Wistar groups, treatment- and gender-specific differences
in infarct size were similar between the two strains. Fig 3 compares infarct size in the cerebral
cortex and caudoputamen among M, F, and O SHR-SP rats. Male
SHR-SP rats sustained larger infarcts compared with females both in the
cerebral cortex (53.1%±4.2% in M versus 35.6%±3.9% in F) and
caudoputamen (53.9%±3.6% in M versus 34.1%±6.5% in
F), a difference that was prevented when female rats were
ovariectomized at an early age (46.5%±4.0% in the cerebral cortex
and 51.4%±4.8% in caudoputamen in O). Fig 4 summarizes averaged residual LD-CBF
over the 2 hours of MCA occlusion in SHR-SP rats. In agreement with an
inverse relation between level of perfusion and the severity of injury,
F rats maintained a higher relative flow during ischemia
compared with M and O rats (18.9%±1.4% in F compared with
11.6%±0.5% and 14.8%±1.5% in M and O, respectively). Fig 5 depicts the correlation between LD-CBF
during ischemia and infarct size in the cerebral cortex, and
demonstrates that the relationship holds true for both sexes and
strains. Fig 6 demonstrates regional
blood flow distribution in representative brain slices
using iodoantipyrine autoradiography at the end of 2
hours of MCA occlusion. In this example, blood flow to MCA territory
was minimally reduced in the ischemic hemisphere in female
brain slices in contrast to the severe reduction in rCBF and formation
of a distinctive core of severely compromised blood flow in
corresponding areas in male and ovariectomized female brain slices. Fig 7 demonstrates the results of point rCBF
measurements for cerebral cortex and caudoputamen in
ischemic and contralateral hemispheres. At the end of
ischemia, female rats sustained higher rCBF in the
caudoputamen compared with M and O rats (62±10 versus
15±7 and 18±6 mL/100 g/min in F, M, and O rats, respectively).
However, mean flow rates in the cerebral cortex were not statistically
different among groups (94±32, 40±17, and 31±7 mL/100 g/min in F, M,
and O rats, respectively). Mean flow rates on the contralateral side
(both the cerebral cortex and caudoputamen) at the end of
ischemia were similar among the three groups (246±70 in M,
237±60 in F, and 213±70 mL/100 g/min in O). In a separate
analysis of cerebrocortical tissue volume at high risk for
infarction, only 11±7 mm3 of the cerebral cortex in F
received less than 10 mL/100 g/min at the end of MCA occlusion compared
with 76±21 mm3 in M. However, a similar level of
ischemic severity was present in 54±21
mm3 of the cerebral cortex in O. This segment of the
cerebral cortex was not statistically different from that of F.
View larger version (23K):
[in a new window]
Figure 1. Cerebral infarct after MCA occlusion in Wistar
rats. MCA was occluded by an intraluminal filament for 2 hours in
age-matched M, F, and O rats. Infarction volumes were measured in brain
sections by TTC staining after 22 hours of reperfusion and expressed as
a percentage of the ipsilateral Ctx or CP. *Significantly different
from M and O groups (P<.05)
View larger version (19K):
[in a new window]
Figure 2. Residual LD-CBF during MCA occlusion in Wistar
rats. LD-CBF was measured over the ipsilateral parietal cerebral cortex
during MCA occlusion in age-matched M, F, and O rats. Control values
were 356±39, 381±24, and 322±32 LD perfusion units in M, F, and O,
respectively. *Significantly different from M and O groups
(P<.05).
View larger version (27K):
[in a new window]
Figure 3. Cerebral infarct after MCA occlusion in SHR-SP.
MCA was occluded by an intraluminal filament for 2 hours in age-matched
M, F, and O rats. Infarction volumes were measured in brain sections by
TTC staining after 22 hours of reperfusion and expressed as a
percentage of the ipsilateral Ctx or CP. *Significantly different from
M and O groups (P<.05).
View larger version (22K):
[in a new window]
Figure 4. Residual LD-CBF during MCA occlusion in SHR-SP.
LD-CBF was measured over the ipsilateral parietal cerebral cortex
during MCA occlusion in age-matched M, F, and O rats. Control values
were 353±27, 381±55, and 416±40 LD perfusion units in M, F, and O,
respectively. *Significantly different from M and O groups
(P<.05).
View larger version (15K):
[in a new window]
Figure 5. Correlation between residual LD-CBF during MCA
occlusion and size of cerebrocortical infarct. LD-CBF was measured in
the ipsilateral parietal cerebral cortex over 2 hours of MCA occlusion
and expressed as percent change from baseline. Infarct size was
measured in TTC-stained brain sections 22 hours after intraluminal
filament occlusion of MCA for 2 hours (r=.79,
P<.001).
View larger version (96K):
[in a new window]
Figure 6. Color-coded distribution of rCBF rates in
representative autoradiograms during
MCA occlusion. Rates of rCBF were measured at the end of 2 hours of
intraluminal filament occlusion of right MCA by [14C]IAP
autoradiography in male, female and ovariectomized
(Ovx) female Wistar rats. Coronal sections correspond to +0.2 (upper
panel) and -1.8 mm (lower panel) relative to the
bregma.
View larger version (21K):
[in a new window]
Figure 7. rCBF rates during MCA occlusion in Wistar rats.
Flow rates were measured by the iodoantipyrine
autoradiographic technique at the end of 2 hours of
unilateral intraluminal filament occlusion of MCA. Flow rates in
representative areas of the cerebral cortex and
caudoputamen were averaged across the ischemic and
nonischemic contralateral hemispheres and compared among M, F,
and O rats. *Significantly different from M and O groups
(P<.05).
Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
The major findings of this study are that (1) female rats sustain
smaller cortical and striatal infarcts after MCA occlusion compared
with age-matched males of both the normotensive Wistar and the SHR-SP
strains, (2) females maintain higher striatal, but not cortical, CBF
than males at the end of vascular occlusion, (3) volume of severely
ischemic tissue (CBF<10 mL/100 g/min) at the end of MCA
occlusion is smaller in the cortex of females compared with males, (4)
gender differences in infarct size are prevented by ovariectomy, which
equalizes plasma estrogen, but not progesterone, in male and female
animals, and (5) ovariectomy eliminates gender differences in
end-ischemic striatal CBF, but does not affect volume of
severely ischemic cortical tissue. These findings provide
evidence for gender-specific responses to cerebrovascular occlusion and
suggest a dual neuroprotective and flow-preserving effect of
endogenous estrogen in the setting of cerebral
ischemia and stroke.
Selected Abbreviations and Acronyms
CBF
=
cerebral blood flow
Ctx
=
cerebral cortex
CP
=
caudoputamen
ER
=
estrogen receptor
F
=
age-matched female rats
IAP
=
[14C]iodoantipyrine
LDF
=
laser-Doppler flowmetric
LD-CBF
=
laser-Doppler flowmetry of cerebral blood flow
M
=
male age-matched rats
MAP
=
mean arterial blood pressure
MCA
=
middle cerebral artery
O
=
age-matched female ovariectomized female rats
rCBF
=
regional cerebral blood flow
SHR-SP
=
stroke-prone spontaneously hypertensive rats
TTC
=
2,3,5-triphenyltetrazolium chloride
Acknowledgments
This study was funded by grants NS3368 and NS20020.
References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
1.
Kannel WB, Thom TJ. The incidence, prevalence and
mortality of cardiovascular disease. In: Schlant RC,
Alexander RW, eds. The Heart. New York, NY: McGraw-Hill,
Inc; 1994:185–197.
Editorial Comment
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
This study fills an important gap in our current stroke
literature. It raises important questions and provides new information
about the influence of gender on brain injury in experimental stroke.
Until recently, there has been little attention to gender difference in
most areas of illness or disability, with the exception of areas that
were specifically gender linked. Consequently, we have limited
information about the influence of gender in most health conditions.
Before 1985 women were rarely included in clinical trials. That
situation has changed in recent years. The National Institutes of
Health has instituted its specific "Guidelines for Inclusion of Women
and Minorities in Clinical Trials," and women are routinely included
in trials.
Selected Abbreviations and Acronyms
CBF
=
cerebral blood flow
Ctx
=
cerebral cortex
CP
=
caudoputamen
ER
=
estrogen receptor
F
=
age-matched female rats
IAP
=
[14C]iodoantipyrine
LDF
=
laser-Doppler flowmetric
LD-CBF
=
laser-Doppler flowmetry of cerebral blood flow
M
=
male age-matched rats
MAP
=
mean arterial blood pressure
MCA
=
middle cerebral artery
O
=
age-matched female ovariectomized female rats
rCBF
=
regional cerebral blood flow
SHR-SP
=
stroke-prone spontaneously hypertensive rats
TTC
=
2,3,5-triphenyltetrazolium chloride
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S.-H. Yang, E. Perez, J. Cutright, R. Liu, Z. He, A. L. Day, and J. W. Simpkins Testosterone increases neurotoxicity of glutamate in vitro and ischemia-reperfusion injury in an animal model J Appl Physiol, January 1, 2002; 92(1): 195 - 201. [Abstract] [Full Text] [PDF]
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T. M. Saleh, A. E. Cribb, and B. J. Connell Reduction in infarct size by local estrogen does not prevent autonomic dysfunction after stroke Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2001; 281(6): R2088 - R2095. [Abstract] [Full Text] [PDF]
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T. M. Saleh, A. E. Cribb, and B. J. Connell Estrogen-induced recovery of autonomic function after middle cerebral artery occlusion in male rats Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2001; 281(5): R1531 - R1539. [Abstract] [Full Text] [PDF]
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N. J. Alkayed, S. Goto, N. Sugo, H.-D. Joh, J. Klaus, B. J. Crain, O. Bernard, R. J. Traystman, and P. D. Hurn Estrogen and Bcl-2: Gene Induction and Effect of Transgene in Experimental Stroke J. Neurosci., October 1, 2001; 21(19): 7543 - 7550. [Abstract] [Full Text] [PDF]
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 H. J. Ranki, G. R. Budas, R. M. Crawford, and A. Jovanovic Gender-specific difference in cardiac ATP-sensitive K+ channels J. Am. Coll. Cardiol., September 1, 2001; 38(3): 906 - 915. [Abstract] [Full Text] [PDF]
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A. Bhardwaj, A. F. Castro III, N. J. Alkayed, P. D. Hurn, and J. R. Kirsch Anesthetic Choice of Halothane Versus Propofol: Impact on Experimental Perioperative Stroke Stroke, August 1, 2001; 32(8): 1920 - 1925. [Abstract] [Full Text] [PDF]
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T. Goyagi, S. Goto, A. Bhardwaj, V. L. Dawson, P. D. Hurn, and J. R. Kirsch Neuroprotective Effect of {{sigma}}1-Receptor Ligand 4-Phenyl-1-(4-Phenylbutyl) Piperidine (PPBP) Is Linked to Reduced Neuronal Nitric Oxide Production Stroke, July 1, 2001; 32(7): 1613 - 1620. [Abstract] [Full Text] [PDF]
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 C. D. Bushnell, G. P. Samsa, and L. B. Goldstein Hormone replacement therapy and ischemic stroke severity in women: A case-control study Neurology, May 22, 2001; 56(10): 1304 - 1307. [Abstract] [Full Text] [PDF]
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 C. W. Hogue Jr, B. Barzilai, K. S. Pieper, L. P. Coombs, E. R. DeLong, N. T. Kouchoukos, and V. G. Davila-Roman Sex Differences in Neurological Outcomes and Mortality After Cardiac Surgery : A Society of Thoracic Surgery National Database Report Circulation, May 1, 2001; 103(17): 2133 - 2137. [Abstract] [Full Text] [PDF]
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V. L. R. Rao, A. Dogan, K. G. Todd, K. K. Bowen, B.-T. Kim, J. D. Rothstein, and R. J. Dempsey Antisense Knockdown of the Glial Glutamate Transporter GLT-1, But Not the Neuronal Glutamate Transporter EAAC1, Exacerbates Transient Focal Cerebral Ischemia-Induced Neuronal Damage in Rat Brain J. Neurosci., March 15, 2001; 21(6): 1876 - 1883. [Abstract] [Full Text] [PDF]
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P. M. Wise, D. B. Dubal, M. E. Wilson, S. W. Rau, and M. Bottner Minireview: Neuroprotective Effects of Estrogen--New Insights into Mechanisms of Action Endocrinology, March 1, 2001; 142(3): 969 - 973. [Abstract] [Full Text] [PDF]
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R. J. Traystman, J. A. Klaus, A. C. DeVries, A. B. Shaivitz, and P. D. Hurn Anticonvulsant Lamotrigine Administered on Reperfusion Fails To Improve Experimental Stroke Outcomes Stroke, March 1, 2001; 32(3): 783 - 787. [Abstract] [Full Text] [PDF]
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L. D. McCullough, N. J. Alkayed, R. J. Traystman, M. J. Williams, and P. D. Hurn Postischemic Estrogen Reduces Hypoperfusion and Secondary Ischemia After Experimental Stroke Stroke, March 1, 2001; 32(3): 796 - 802. [Abstract] [Full Text] [PDF]
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D. B. Dubal and P. M. Wise Neuroprotective Effects of Estradiol in Middle-Aged Female Rats Endocrinology, January 1, 2001; 142(1): 43 - 48. [Abstract] [Full Text] [PDF]
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J. Krejza, Z. Mariak, M. Huba, S. Wolczynski, and J. Lewko Effect of Endogenous Estrogen on Blood Flow Through Carotid Arteries Stroke, January 1, 2001; 32(1): 30 - 36. [Abstract] [Full Text] [PDF]
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M. I. Rossberg, S. J. Murphy, R. J. Traystman, P. D. Hurn, and H. A. Kontos LY353381.HCl, a Selective Estrogen Receptor Modulator, and Experimental Stroke Editorial Comment Stroke, December 1, 2000; 31(12): 3041 - 3046. [Abstract] [Full Text] [PDF]
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R. S. Briellmann, S. F. Berkovic, and G. D. Jackson Men may be more vulnerable to seizure-associated brain damage Neurology, November 28, 2000; 55(10): 1479 - 1485. [Abstract] [Full Text] [PDF]
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K. Sampei, A. S. Mandir, Y. Asano, P. C. Wong, R. J. Traystman, V. L. Dawson, T. M. Dawson, P. D. Hurn, and C. Y. Hsu Stroke Outcome in Double-Mutant Antioxidant Transgenic Mice Editorial Comment Stroke, November 1, 2000; 31(11): 2685 - 2691. [Abstract] [Full Text] [PDF]
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T. K. Toung, P. D. Hurn, R. J. Traystman, F. E. Sieber, and F. M. Faraci Estrogen Decreases Infarct Size After Temporary Focal Ischemia in a Genetic Model of Type 1 Diabetes Mellitus Editorial Comment Stroke, November 1, 2000; 31(11): 2701 - 2706. [Abstract] [Full Text] [PDF]
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P. M. Wise and D. B. Dubal Estradiol Protects Against Ischemic Brain Injury in Middle-Aged Rats Biol Reprod, October 1, 2000; 63(4): 982 - 985. [Abstract] [Full Text]
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M. T. Littleton-Kearney, D. M. Agnew, R. J. Traystman, and P. D. Hurn Effects of estrogen on cerebral blood flow and pial microvasculature in rabbits Am J Physiol Heart Circ Physiol, September 1, 2000; 279(3): H1208 - H1214. [Abstract] [Full Text] [PDF]
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G. G. Geary, D. N. Krause, and S. P. Duckles Estrogen reduces mouse cerebral artery tone through endothelial NOS- and cyclooxygenase-dependent mechanisms Am J Physiol Heart Circ Physiol, August 1, 2000; 279(2): H511 - H519. [Abstract] [Full Text] [PDF]
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A. Bhardwaj, I. Harukuni, S. J. Murphy, N. J. Alkayed, B. J. Crain, R. C. Koehler, P. D. Hurn, R. J. Traystman, and J. C. Watson Hypertonic Saline Worsens Infarct Volume After Transient Focal Ischemia in Rats Editorial Comment Stroke, July 1, 2000; 31(7): 1694 - 1701. [Abstract] [Full Text] [PDF]
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S. J. Murphy, R. J. Traystman, P. D. Hurn, and S. P. Duckles Progesterone Exacerbates Striatal Stroke Injury in Progesterone-Deficient Female Animals Editorial Comment Stroke, May 1, 2000; 31(5): 1173 - 1178. [Abstract] [Full Text] [PDF]
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I. Harukuni, A. Bhardwaj, A. B. Shaivitz, A. C. DeVries, E. D. London, P. D. Hurn, R. J. Traystman, J. R. Kirsch, and F. M. Faraci {sigma}1-Receptor Ligand 4-Phenyl-1-(4-Phenylbutyl)-Piperidine Affords Neuroprotection From Focal Ischemia With Prolonged Reperfusion • Editorial Comment Stroke, April 1, 2000; 31(4): 976 - 982. [Abstract] [Full Text] [PDF]
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K. Sampei, S. Goto, N. J. Alkayed, B. J. Crain, K. S. Korach, R. J. Traystman, G. E. Demas, R. J. Nelson, P. D. Hurn, and S. Piper Duckles Stroke in Estrogen Receptor-{alpha}-Deficient Mice • Editorial Comment Stroke, March 1, 2000; 31(3): 738 - 744. [Abstract] [Full Text] [PDF]
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S.-H. Yang, J. Shi, A. L. Day, J. W. Simpkins, and S. E. Robinson Estradiol Exerts Neuroprotective Effects When Administered After Ischemic Insult • Editorial Comment Stroke, March 1, 2000; 31(3): 745 - 750. [Abstract] [Full Text] [PDF]
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K. Fukuda, H. Yao, S. Ibayashi, T. Nakahara, H. Uchimura, M. Fujishima, and E. D. Hall Ovariectomy Exacerbates and Estrogen Replacement Attenuates Photothrombotic Focal Ischemic Brain Injury in Rats Editorial Comment Stroke, January 1, 2000; 31(1): 155 - 160. [Abstract] [Full Text] [PDF]
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N. J. Alkayed, S. J. Murphy, R. J. Traystman, P. D. Hurn, and V. M. Miller Neuroprotective Effects of Female Gonadal Steroids in Reproductively Senescent Female Rats Editorial Comment Stroke, January 1, 2000; 31(1): 161 - 168. [Abstract] [Full Text] [PDF]
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H. V. O. Carswell, A. F. Dominiczak, and I. M. Macrae Estrogen status affects sensitivity to focal cerebral ischemia in stroke-prone spontaneously hypertensive rats Am J Physiol Heart Circ Physiol, January 1, 2000; 278(1): H290 - H294. [Abstract] [Full Text] [PDF]
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Recommendations for Standards Regarding Preclinical Neuroprotective and Restorative Drug Development Stroke, December 1, 1999; 30(12): 2752 - 2758. [Abstract] [Full Text] [PDF]
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A. M. McNeill, N. Kim, S. P. Duckles, D. N. Krause, and H. A. Kontos Chronic Estrogen Treatment Increases Levels of Endothelial Nitric Oxide Synthase Protein in Rat Cerebral Microvessels • Editorial Comment Stroke, October 1, 1999; 30(10): 2186 - 2190. [Abstract] [Full Text] [PDF]
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D. B. Dubal, P. J. Shughrue, M. E. Wilson, I. Merchenthaler, and P. M. Wise Estradiol Modulates bcl-2 in Cerebral Ischemia: A Potential Role for Estrogen Receptors J. Neurosci., August 1, 1999; 19(15): 6385 - 6393. [Abstract] [Full Text] [PDF]
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R. Rusa, N. J. Alkayed, B. J. Crain, R. J. Traystman, A. S. Kimes, E. D. London, J. A. Klaus, P. D. Hurn, and C. Iadecola 17{beta}-Estradiol Reduces Stroke Injury in Estrogen-Deficient Female Animals • Editorial Comment Stroke, August 1, 1999; 30(8): 1665 - 1670. [Abstract] [Full Text] [PDF]
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P. M. Wise, M. J. Smith, D. B. Dubal, M. E. Wilson, K. M. Krajnak, and K. L. Rosewell Neuroendocrine Influences and Repercussions of the Menopause Endocr. Rev., June 1, 1999; 20(3): 243 - 248. [Abstract] [Full Text]
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K. Kasischke, R. Huber, H. Li, M. Timmler, M. W. Riepe, and D. O. Carpenter Primary Hypoxic Tolerance and Chemical Preconditioning During Estrus Cycle in Mice • Editorial Comment Stroke, June 1, 1999; 30(6): 1256 - 1262. [Abstract] [Full Text] [PDF]
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T. J. Toung, A. Bhardwaj, V. L. Dawson, T. M. Dawson, R. J. Traystman, P. D. Hurn, and P. H. Chan Neuroprotective FK506 Does Not Alter In Vivo Nitric Oxide Production During Ischemia and Early Reperfusion in Rats • Editorial Comment Stroke, June 1, 1999; 30(6): 1279 - 1285. [Abstract] [Full Text] [PDF]
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Q. Wang, R. Santizo, V. L. Baughman, D. A. Pelligrino, and C. Iadecola Estrogen Provides Neuroprotection in Transient Forebrain Ischemia Through Perfusion-Independent Mechanisms in Rats • Editorial Comment Stroke, March 1, 1999; 30(3): 630 - 637. [Abstract] [Full Text] [PDF]
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H. V. O. Carswell, N. H. Anderson, J. S. Clark, D. Graham, B. Jeffs, A. F. Dominiczak, and I. M. Macrae Genetic and Gender Influences on Sensitivity to Focal Cerebral Ischemia in the Stroke-Prone Spontaneously Hypertensive Rat Hypertension, February 1, 1999; 33(2): 681 - 685. [Abstract] [Full Text] [PDF]
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H. Cai, H. Yao, S. Ibayashi, H. Uchimura, M. Fujishima, and B. D. Watson Photothrombotic Middle Cerebral Artery Occlusion in Spontaneously Hypertensive Rats: Influence of Substrain, Gender, and Distal Middle Cerebral Artery Patterns on Infarct Size • Editorial Comment Stroke, September 1, 1998; 29(9): 1982 - 1987. [Abstract] [Full Text] [PDF]
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T. J. K. Toung, R. J. Traystman, P. D. Hurn, and V. M. Miller Estrogen-Mediated Neuroprotection After Experimental Stroke in Male Rats • Editorial Comment Stroke, August 1, 1998; 29(8): 1666 - 1670. [Abstract] [Full Text] [PDF]
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