Thomas K. Toung, Patricia D. Hurn, Richard J. Traystman and Frederick E. Sieber
Type 1 Diabetes Mellitus
Estrogen Decreases Infarct Size After Temporary Focal Ischemia in a Genetic Model of
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Estrogen Decreases Infarct Size After Temporary Focal
Ischemia in a Genetic Model of Type 1 Diabetes Mellitus
Thomas K. Toung, MD; Patricia D. Hurn, PhD; Richard J. Traystman, PhD; Frederick E. Sieber, MD
Background and Purpose—It is unclear how genetic type 1 diabetes mellitus (DM) influences infarct size when blood
glucose is tightly controlled. The aim of this study was to determine the effect of genetic type 1 DM, as occurs in BB
rats, on infarct size after transient unilateral middle cerebral artery occlusion (MCAO) in male and female rats. In
addition, studies suggest that male type 1 DM rats have a higher incidence of end-organ complications than do females.
A second aim of this study was to determine the effect of chronic 17?-estradiol (E2) administration on infarct size in
male BB rats.
Methods—Diabetic male (MDiab, n?14) and female (FDiab, n?8) BB rats were studied and compared with background
strain Wistar rats (MWist, n?16; FWist, n?14). Two additional male cohorts (MWist?E2, n?15; MDiab?E2, n?14)
received subcutaneous 25 ?g E2implants 7 to 10 days before MCAO. Rats underwent 1 hour of MCAO followed by
22 hours of reperfusion. Physiological variables were controlled among groups, and the intraischemic laser Doppler flow
signal was reduced similarly in all animals. Infarction volume was evaluated by 2,3,5-triphenyltetrazolium chloride
staining and image analysis.
Results—Preischemic blood glucose was 94?5, 127?13, 90?15, 63?18, 122?8, and 81?14 mg/dL in MWist, FWist,
MDiab, FDiab, MWist?E2, and MDiab?E2rats, respectively (mean?SE). Intraischemic laser Doppler flow was
reduced to 20% to 25% of baseline in all groups. Striatal infarct size (percentage of ipsilateral caudate putamen) was
increased in male diabetic rats relative to nondiabetic MWist rats (41?3% versus 28?3%). Striatal injury was not
increased in FDiab rats, and infarction volume was smaller than that in FWist rats (23?4% in FWist versus 13?3% in
FDiab). Chronic estrogen treatment reduced cortical and striatal infarction in MDiab?E2rats compared with untreated
Conclusions—Type 1 DM is associated with increased infarct size after temporary MCAO, despite tight control of blood
glucose. The deleterious effect of DM is evident only in males rats; female diabetic BB rats sustain small infarcts.
Chronic E2treatment reduced injury in the male BB rat, providing neuroprotection even in the presence of DM. These
data suggest that genetic diabetes even with mild glucose elevation plays a role in determining neuropathology in
experimental stroke. However, factors such as reproductive steroids also determine outcome in DM stroke. (Stroke.
Key Words: brain?cerebral ischemia?complications?diabetes mellitus?estrogen?ischemia
?middle cerebral artery?stroke?rats
controlled. This issue is important because DM is an inde-
pendent risk factor for stroke.1In addition, DM is strongly
related to early stroke progression and associated with poorer
stroke outcome.2Transient ischemia, whether global or focal,
is associated with greater neuropathologic damage in DM
hyperglycemic animals. In particular, the preischemic blood
glucose is an important determinant of neurological outcome
after temporary ischemia in DM.3Insulin-based blood glu-
cose management has been shown to decrease infarct size.
However, it is still unresolved whether DM, independent of
t is unclear how genetic type 1 diabetes mellitus (DM)
influences infarct size when blood glucose is tightly
See Editorial Comment, page 2706
the blood glucose level, affects infarct size after temporary
brain ischemia. The aim of the present study was to determine
the effect of genetic type 1 DM, as occurs in male and female
BB rats, on infarct size after transient middle cerebral artery
Many studies have shown that supplementary estrogen
administration is neuroprotective in stroke. Studies examin-
ing the epidemiology of DM have shown that male type 1
DM rats have a higher incidence of end-organ complications
than do female rats.4A second aim of the present study was
Received April 7, 2000; final revision received August 7, 2000; accepted August 8, 2000.
From the Department of Anesthesiology and Critical Care Medicine, Johns Hopkins Medical Institutions, and the Department of Anesthesiology
(F.E.S.), Johns Hopkins Bayview Medical Center, Baltimore, Md.
Correspondence to Frederick E. Sieber, MD, Vice Chairman, Department of Anesthesiology, Johns Hopkins Bayview Medical Center, 4940 Eastern
Ave, Baltimore, MD 21224-2780. E-mail firstname.lastname@example.org
© 2000 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org
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to determine whether chronic preischemic estrogen treatment
reduced infarction size in male type 1 DM rats treated with
Materials and Methods
The present study was conducted in accordance with the National
Institutes of Health guidelines for the care and use of animals in
research. All protocols were approved by the Animal Care and Use
Committee of the Johns Hopkins University. All methods are as
Diabetic Animal Model
Studies were conducted in type 1 DM male and female BB rats
(Biobreeding Laboratories, Ottawa, Ontario, Canada) and in age-
matched background strain Wistar rats of both sexes. BB rats were
obtained from the supplier after ?5 weeks of DM and then were
housed for 1 to 3 weeks while receiving intensive insulin therapy
with a goal of maintaining blood glucose ?150 mg/dL. Additional
groups of male BB and Wistar rats received subcutaneous implants
of 17?-estradiol (E2, 25 ?g) ?7 to 10 days before MCAO, as
previously described.5,6The 25-?g implant treatment was chosen on
the basis of previous efficacy in reducing stroke injury after MCAO
in male,6female,7and reproductively senescent female Wistar rats.8
One week of treatment with the implant yields a stable and
physiological level of plasma E2(ie, 10 to 20 pg/mL).5–8Animals
were housed with free access to water and food, and subcutaneous
insulin was administered daily in response to blood glucose levels
obtained from tail samples.
Reversible Unilateral MCAO
Male and female rats were anesthetized with 1% to 2% halothane
delivered via face mask in oxygen-enriched air and instrumented
with femoral artery catheters for physiological monitoring and blood
gas measurement. Rectal and temporalis muscle temperatures were
controlled at 37.5?0.5°C by use of heat lamps. Cortical perfusion
was measured by laser Doppler flowmetry (LDF, Moor Instruments
Ltd, model MBF3D) as previously described, with probe placement
at 2 mm posterior and 6 mm lateral to the bregma. Unilateral focal
cerebral ischemia was accomplished by use of the intraluminal
filament model (4-0 nylon monofilament suture) of proximal
MCAO. The right common carotid artery was exposed through a
lateral incision, separated from the vagus, and ligated. The external
carotid artery was ligated, the occipital branch was cauterized, and
the pterygopalatine artery was ligated. An occluding filament was
advanced via the common carotid artery until the LDF signal
displayed an abrupt and significant reduction, confirming ongoing
ischemia; it was then secured in place. Ischemic LDF was deter-
mined over 5-minute periods throughout the 60-minute occlusion
period, and then the suture was withdrawn with prompt restoration of
blood flow. Each animal was recovered and supported with intrave-
nous Ringer’s lactate solution and supplemental oxygen as needed.
Insulin was given after surgery to maintain plasma glucose at the
target level. After 22 hours of reperfusion, the animal was reanes-
thetized for harvesting of the brain. The tissue was sliced into seven
2-mm-thick coronal sections for 2,3,5-triphenyltetrazolium chloride
staining and quantification via standard photography and digital
planimetry (SigmaScan Pro, Jandel). The infarcted area was numer-
ically integrated across each section and over the entire ipsilateral
hemisphere. Infarct volume was measured separately in the cortex
and caudate putamen and expressed as a volume percentage of the
All values are reported as mean?SE; all physiological variables,
infarct volumes, and residual LDF were analyzed by 1-way ANOVA
and post hoc Newman-Keuls test. The relationship between preis-
chemic glucose and infarction was analyzed by Pearson correlation.
Statistical significance was confirmed at value of P? 0.05.
Six groups were studied: nondiabetic male (n?16, mean body
weight 310?15 g) and female (n?14, 262?13 g) Wistar rats,
diabetic male (n?14, mean body weight 355?7 g) and
female (n?8, mean body weight 299?5 g) BB rats, and
E2-treated male Wistar (n?15, mean body weight 341?10 g)
and BB (n?14, mean body weight 373?7 g) rats. Physio-
logical variables were controlled during the peri-ischemic
period, as shown in the Table. Preischemic glucose was
higher in diabetic males than in females; otherwise, there
MAP, mm Hg
PaCO2, mm Hg
PaO2, mm Hg
Values are mean?SE for baseline and at 60 minutes of MCAO. Arterial glucose was measured
immediately before ischemia and on reperfusion. MAP indicates means arterial blood pressure; PaCO2,
arterial carbon dioxide tension; PaO2, arterial oxygen tension; MWist, male nondiabetic Wistar rats;
FWist, female nondiabetic Wistar rats; MDiab, male diabetic BB strain; FDiab, female diabetic BB
strain; MWistE2, E2-treated male Wistar rats (25 ?g implant); and MDiabE2, E2-treated male diabetic
BB strain (25 ?g implant).
*P?0.05 vs male strain-matched group.
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were no baseline differences among treatment groups. There
was no difference in intraischemic reduction of the LDF
signal among groups (Figure 1).
Figure 2 displays infarction volumes for the 2 brain regions
damaged by 1 hour of MCAO, the cortex and caudate
putamen, as a percentage of the respective ipsilateral struc-
ture. Cortical injury was small in all treatment groups,
particularly in diabetic females, in which no injury was
present, and in female nondiabetic Wistar rats (Figure 2). In
the striatum, injury volumes were larger, and differences
linked to diabetic strain and to sex were apparent. Diabetic
males sustained larger striatal infarction compared with their
male Wistar counterparts (Figure 2). Diabetes-linked exacer-
bation of stroke injury was sex dependent in that female
diabetic rats did not demonstrate increased striatal injury.
Furthermore, striatal infarction was surprisingly smaller in
diabetic BB than in Wistar females.
Figure 3 shows the effect of estrogen treatment in male
diabetic and Wistar rats compared with their untreated male
counterparts in the same strain. When untreated and E2-
treated Wistar males were compared, E2treatment did not
alter the relatively small damage sustained after 60 minutes of
MCAO in the cortex or caudate putamen. However, when
untreated and E2-treated male diabetic BB rats were com-
pared, E2clearly produced a robust protection in both the
cortex and striatum.
Figure 4 plots individual animal values for preischemic
plasma glucose concentration and striatal infarction within all
experimental groups. There was no strong correlation be-
tween these variables (r?0.191), in part because hyperglyce-
mia was limited by the study design. Therefore, only limited
ranges of glucose were present (?200 mg/dL), and most
animals fell in the 75- to 150-mg/dL range. The correlation
was not improved by comparison by sex or estrogen treat-
ment. Although many females or estrogen-treated males had
normal or low preischemic glucose levels, others sustained
high glucose levels but small infarctions.
The present study shows that type 1 DM is associated with
increased infarct size after temporary MCAO, despite tight
control of blood glucose. The deleterious effect of DM is
evident only in males, whereas female diabetic BB rats
sustain small infarcts. Chronic E2treatment reduced injury in
the male BB rat, providing neuroprotection even in the
presence of DM. These data suggest that genetic DM even
with mild glucose elevation plays a role in determining
neuropathology in experimental stroke. However, factors
such as reproductive steroids also determine outcome in DM
Figure 1. Ipsilateral parietal LDF during MCAO and reperfusion
in the 6 treatment groups. Reduction in ischemic LDF was not
different among groups. Rat groups are as follows: male Wistar,
n?16; male diabetic BB strain, n?14; female Wistar, n?14;
female diabetic BB strain , n?8; male Wistar with subcutaneous
E2implant (MaleWist?E2), n?15; and male diabetic with E2
implant (MaleDiab?E2), n?14.
Figure 2. Effect of diabetes on outcome from MCAO in male
and female rats. Cortical and caudate putamen infarction vol-
ume is shown as percentage of ipsilateral (ipsi) structure. Rat
groups are as follows: male Wistar, n?16; male diabetic BB
strain, n?14; female Wistar, n?14; and female diabetic BB
strain, n?8. *P?0.05 vs Wistar strain of matching sex.
Figure 3. Effect of E2treatment in male diabetic rats. Cortical
and caudate putamen infarction volume is shown as percentage
of ipsilateral (ipsi) structure. Rat groups are as follows: male
Wistar (no-Rx Wistar), n?16; male diabetic BB strain (no-Rx dia-
betic, n?14); male Wistar with subcutaneous E2implant (E2-Rx
Wistar), n?15; and male diabetic with E2implant (E2-Rx diabet-
ic), n?14. *P?0.05 vs untreated male diabetic animals.
Toung et alEstrogen Decreases Infarct Size in DM
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Transient ischemia, whether global or focal, is associated
with greater neuropathologic damage in DM hyperglycemic
animals. During temporary focal ischemia, DM hyperglyce-
mia increases infarct size.9Similarly, with transient global
ischemia, the level of DM hyperglycemia also affects the
neurological outcome. In the rat forebrain ischemia model,
preischemic treatment of DM hyperglycemia with insulin
leads to a neurological outcome and histopathology similar to
that in non-DM normoglycemic rats.10However, untreated
hyperglycemia leads to a dismal neurological and histopatho-
logic outcome. These results agree with studies using mag-
netic resonance spectroscopy, which suggest that the most
hyperglycemic DM animals are at highest risk for poor
neurological outcome.11Similarly, in dogs, the preischemic
blood glucose level is an important determinant of morbidity
and neuropathologic damage after global brain ischemia in
insulin-dependent animals.12It is unclear whether the above-
mentioned results represent a neuroprotective effect of insulin
or a neurotoxic effect of hyperglycemia. However, they
emphasize the potential importance of insulin-based blood
glucose control in determining neurological outcome after
transient brain ischemic insults in DM. In the present study,
intensive insulin-based blood glucose management was un-
dertaken 1 to 3 weeks before MCAO to eliminate hypergly-
cemic effects. We targeted blood glucose at ?200 mg/dL
immediately before the onset of MCAO, and preischemic
glucose values were similar in the Wistar and diabetic male
groups. Despite this insulin-intensive management of blood
glucose, ischemic injury was greater in type 1 DM males.
Previous studies used chemical or surgically induced DM.
These animal models may not accurately reflect type 1 DM,
which entails complex genetic and immunologic interactions.
In the present study, a genetic rodent model of type 1 DM was
used, simulating some complexity of human DM. There are
several reasons to suspect that DM, even with intensive
insulin-based blood glucose management, would be associ-
ated with worsened neurological outcome after stroke. In
particular, DM alters cerebral blood flow and reactivity.3
These effects have been observed in larger vessels and in the
microvasculature.13Autopsy studies suggest that diabetics are
particularly prone to cerebral small-artery disease, lacunar
infarction, and large-artery atherosclerotic occlusive dis-
ease.14DM-induced vascular abnormalities may have rele-
vance to the present results with the present model of MCAO,
which would simulate this latter pathology. Although the
reduction of LDF during vascular occlusion was similar
among all groups in the study (?20% to 25% of baseline
signal), absolute intraischemic cerebral blood flow was not
measured. If type 1 DM altered baseline cerebral blood flow,
then differences in intraischemic blood flow cannot be
excluded and may have influenced tissue outcome. Last, in
our 1-hour MCAO model, the effect of DM was most striking
in the striatum, which contains the core of the ischemic
lesion, rather than the cortex. A moderate intensity of
ischemic duration was chosen to best ensure survivorship
within the insulin-dependent diabetic cohort. Consequently,
cortical injury levels were small in all treatment groups
within the study and may have been somewhat insensitive to
the effects of genetic strain or treatment. Alternatively,
genetic diabetes may recruit potentially salvageable tissue
into the core of the lesion by compromising collateral
circulation and vasodilatory capacity during MCAO.
The present data strongly suggest that sex and estrogen
availability have large influences on the outcome from stroke
in the diabetic strain. Although DM exacerbates stroke
damage in the male, the female is spared. Our laboratory5and
others15–17have demonstrated that female animals sustain
reduced tissue damage from experimental stroke; loss of
endogenous reproductive steroids in females results in ische-
mic damage, which becomes indistinguishable from that of
age-matched males. Furthermore, exogenous estrogen treat-
ment has been widely shown to be neuroprotective in ische-
mic injury both in vivo and in vitro (for review, see Reference
18). The anti-ischemic mechanism(s) of the steroid is under
heavy investigation18; however, both vascular5,19–21and non-
vascular7,22,23sites of action have been demonstrated. Cellular
mechanisms are likely multifactorial, including preservation
of vascular tone and residual cerebral blood flow during
cerebral ischemia,5,20,21induction of protective gene prod-
ucts,24antioxidant activity,15,25modification of inflammatory
processes,26and reduction of glutamate excitotoxicity.27The
present data further these observations by demonstrating that
genetically diabetic female rodents are also protected from
ischemic damage relative to the male of the same strain and
that estrogen treatment strongly protects the male diabetic
brain during MCAO. Estrogen implants reduced infarction
volume in the diabetic brain to levels not different from those
observed in the nondiabetic brain. These findings emphasize
that outcome from DM stroke, like non-DM stroke, is sex
dependent. Furthermore, the deleterious effect of DM in the
Figure 4. Lack of relationship between plasma glucose sam-
pled immediately before to occlusion and ipsilateral (ipsi) striatal
infarction volume. Data are compiled from individual animals
within all treatment groups. Sex or estrogen treatment was not
associated with selective reduction of plasma glucose and
decreased infarction. Rat groups are as follows: male Wistar
with and without E2treatment (MWist and MWistE2, respective-
ly), n?16 and 15, respectively; male diabetic BB strain with or
without E2treatment (MDiab and MDiabE2, respectively), n?14
and 14, respectively; female Wistar (FWist), n?14; and female
diabetic BB strain (FDiab), n?8.
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male brain confronted with an ischemic stress is responsive to
a neuroprotectant, such as estrogen.
The importance of sex or reproductive steroids in type 1
DM end-organ complications has not been extensively stud-
ied in human or animal models. Although premenopausal
nondiabetic women have lower stroke rates than age-matched
males (for review, see Reference 18), this relationship has not
been specifically studied in diabetic women. Diabetes is a
known risk factor for stroke in both men and women, but
most epidemiological studies have not reported sex-specific
relative risks. Whether estrogen treatment alters stroke out-
come in human DM is also unclear; however, data from the
Framingham study show that stroke damage is greatly in-
creased in postmenopausal diabetic versus nondiabetic wom-
en.28Several factors in the present rodent experiments must
be considered relative to the neuroprotection of estrogen.
First, because preischemic plasma glucose levels were, on
average, lower in DM females than in males, we cannot
exclude this factor as a source of the protection enjoyed by
the female. Preischemic hyperglycemia is known to increase
brain damage by fueling lactic acidosis and depressing
intraischemic tissue pH,12,13,29potentially enhancing oxygen
radical–mediated injury mechanisms.29,30However, glucose
levels were relatively controlled in all the BB rats for 1 to 3
weeks before MCAO. The level of hyperglycemia present in
any of the animals was mild and consequently not well
correlated with increased infarction for males or females.
Second, the observation that striatal damage in diabetic
females was less severe than in nondiabetic females could
suggest an overlap of tissue mechanisms by which estrogen
protects brain and DM increases stroke damage. This is the
first report of sex differences in DM stroke or of the efficacy
of estrogen in reducing injury in the DM male, so further
studies are needed to determine cellular mechanisms of
interaction. Potential interactions between estrogen and DM
could involve the ability of the steroid to enhance cerebral
perfusion under conditions of vascular stress,5,19,20protect
vascular endothelial integrity,19enhance glucose transport
and increase blood-brain barrier expression of the glucose
transporter GLUT1,31–33and decrease oxidative impairment
of membrane Na?,K?-ATPase activity.34Last, estrogen treat-
ment did not decrease infarction volume in nondiabetic male
rats, contrary to previous reports of significant efficacy in the
male brain.6,34The lack of neuroprotection is likely related to
the small degree of injury produced in nondiabetic animals
after a single hour of MCAO. When the duration of MCAO
is extended and larger tissue volumes are compromised,
chronic and acute estrogen treatment ameliorates focal injury
in the nondiabetic brain.6,34
In conclusion, genetic type 1 DM negatively influences
infarct size when hyperglycemia is tightly controlled. This
effect is limited to the male and can be altered by estrogen
availability, suggesting that these factors be considered in
further studies of DM stroke. Vascular and parenchymal
mechanisms by which DM enhances stroke damage require
This study was supported by American Heart Association Grant-in-
Aid MDSG5597 and National Institutes of Health grants NS-33668,
NS-20020, and NR-03521
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Diabetes mellitus increases the risk for stroke. The goal of
this study was to examine effects of genetic, type 1 diabetes
on infarct size in a rat model of focal cerebral ischemia in
which relatively good control of plasma glucose levels were
achieved by using insulin. In addition, the study examined the
influence of chronic treatment with 17?-estradiol on infarct
size during diabetes.
The results suggest that type 1 diabetes is associated with
increased infarct size, even with relatively good control of
plasma glucose levels. This increase in brain injury in
response to ischemia was seen in males and could be reduced
by 17?-estradiol. Thus, these findings support the concept
that estrogen is neuroprotective. In contrast to the results in
males, females rats with diabetes did not have increased
infarct size following ischemia.
At the present time, it is unclear what mechanism(s)
produce gender-specific increases in brain injury following
ischemia during diabetes. What are some possibilities? There
are several levels at which one might hypothesize that
diabetes could exacerbate brain injury following ischemia.
These include diabetes-induced vascular dysfunction and
reductions in blood flow, increases in production of reactive
oxygen species (or reductions in effectiveness of antioxidant
mechanisms), or reprogramming of gene expression such that
subsequent injury in response to ischemia is enhanced.
Although it is fairly well established that estrogen has
neuroprotective effects in cerebral ischemia, the present
work provides new evidence that this protective effect
extends to the setting of diabetes. The mechanisms that
account for neuroprotection during diabetes also remain to
Frank M. Faraci, PhD, Guest Editor
Department of Internal Medicine
University of Iowa College of Medicine
Iowa City, Iowa
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