Hyperglycemia Exacerbates Brain Edema and

Perihematomal Cell Death After Intracerebral Hemorrhage

Eun-Chol Song, MD; Kon Chu, MD; Sang-Wuk Jeong, MD; Keun-Hwa Jung, MD;
Seong-Hoon Kim, MD, PhD; Manho Kim, MD, PhD; Byung-Woo Yoon, MD, PhD

Background and Purpose—Hyperglycemia has a deleterious effect on brain ischemia. However, the effect of
hyperglycemia in intracerebral hemorrhage (ICH) is not well known. We investigated the effect of hyperglycemia on
the development of brain edema and perihematomal cell death in ICH.
Methods—Hyperglycemia was induced by intraperitoneal injection of streptozotocin (60 mg/kg) in adult Sprague-Dawley
male rats. ICH was induced by stereotaxic infusion of 0.23 U of collagenase into the left striatum. Seventy-two hours
after ICH, terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end labeling (TUNEL) staining was
performed for perihematomal cell death. We also measured brain water content to evaluate edema formation.
Results—The serum glucose level of the hyperglycemic group was 394.0⫾180.3 mg/dL (n⫽31), and that of the
normoglycemic group was 97.5⫾27.4 mg/dL (n⫽31). The size of hemorrhage was similar between groups, without any
significant difference (n⫽8 in each group). The brain water content of hyperglycemic rats (n⫽17) increased in both
lesioned (81.0⫾0.5%) and nonlesioned hemispheres (78.7⫾0.6%) compared with the normoglycemic group (n⫽17;
lesioned: 78.9⫾0.8%; nonlesioned: 77.3⫾1.1%). In the hyperglycemic group, more TUNEL-positive cells were found
in the perihematomal regions (n⫽6).
Conclusions—Hyperglycemia caused more profound brain edema and perihematomal cell death in experimental ICH.
(Stroke. 2003;34:2215-2220.)
Key Words: brain edema 䡲 hyperglycemia 䡲 intracerebral hemorrhage 䡲 neuronal death 䡲 streptozotocin

H yperglycemia has been associated with increased neu- because of hematoma expansion or large hematoma and brain
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ronal damage in animal models of ischemia.1,2 Hyper-
glycemic or diabetic patients with stroke tend to have a
worsened neurological outcome and an increased hemor-
rhagic transformation rate.3,4 Animal experiments of cerebral
ischemia have elucidated mechanisms of the deleterious
effects of hyperglycemia on the damaged brain, which
include acidosis, release of excitatory amino acids, exagger-
ation of edema formation, blood-brain barrier (BBB) injury,
and hemorrhagic transformation of the infarct.1,5,6
Intracerebral hemorrhage (ICH) represents at least 15% of
all strokes in Western populations7 and a considerably higher
proportion (20% to 30%) in Asian and black populations.8,9
The prognosis of ICH is poor, often much worse than that of
ischemic strokes of similar size.9 Patients with ischemic
stroke (up to 30%) will undergo hemorrhagic transforma-
tion,10 and a considerable number of ischemic infarct patients
after thrombolysis develop hemorrhagic conversion or symp-
tomatic hematoma.11 Brain edema is also usually associated
with deterioration in ICH. Many patients can deteriorate
edema development.9,12–14 However, the exact mechanism of
edema formation after ICH is still unknown. Moreover, there
is no animal study showing the effect of hyperglycemia on
the brain edema associated with ICH.
To investigate the pathogenesis of ICH, various animal
models have been developed.15–17 In the present study we
examined the hypothesis that hyperglycemia may exert its
effect on the formation of brain edema and associated
perihematomal cell death after ICH.
Materials and Methods
Induction of Hyperglycemia and ICH
All the procedures were performed following an institutionally
approved protocol in accordance with the National Institutes of
Health Guide for the Care and Use of Laboratory Animals. Sixty-
two male Sprague-Dawley rats (Osan, Samtako) weighing 240 to
280 g were used. Rats were grouped as hyperglycemic (n⫽31) and
normoglycemic (n⫽31). Hyperglycemia was induced by intraperito-
neal injection of streptozotocin (60 mg/kg; Sigma) 3 days before
operation.18 Experimental ICH was induced by the stereotaxic

Received January 21, 2003; final revision received May 9, 2003; accepted May 28, 2003.
From the Department of Neurology and Clinical Research Institute, Seoul National University Hospital, Seoul (E-C.S., K.C., K-H.J., M.K., B-W.Y.);
Neuroscience Research Institute of Seoul National University Medical Research Center, Seoul (E-C.S., K.C., K-H.J., M.K., B-W.Y.); Department of
Neurology, Seoul National Hospital, Seoul (K.C.); Department of Neurology, Ilsan Paik Hospital, Inje University, Ilsan (S-W.J.); and Department of
Neurology, Gangwon University, Chunchon (S-H.K.), South Korea. Dr Song and Dr Chu contributed equally to this work.
Reprint requests to Byung-Woo Yoon, MD, PhD, Department of Neurology, Seoul National University Hospital, 28 Yongon-dong, Chongno-gu, Seoul
110-744, South Korea. E-mail bwyoon@snu.ac.kr
© 2003 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org DOI: 10.1161/01.STR.0000088060.83709.2C

2215
 2216 Stroke September 2003
Figure 1. Mean serum glucose level after ICH induction. Values
are mean⫾SD. *P⬍0.01 compared with normoglycemic group.
intrastriatal administration of bacterial collagenase type IV (Sigma).
In brief, after an intraperitoneal injection of 1% ketamine (30 mg/kg;
Sigma) and xylazine hydrochloride (4 mg/kg; Sigma), rats were
placed in a stereotaxic frame (David Kopf Instruments). A burr hole
was made, and a 30-gauge Hamilton syringe needle was inserted into
the striatum (location: 3.0 mm left lateral to the midline, 0.2 mm
posterior to bregma, 6 mm in depth below the skull).19 ICH was
induced by the administration of 1 ␮L containing 0.23 collagen
digestion unit of collagenase type IV (Sigma) over 5 minutes. After
completion of collagenase infusion, the burr hole was sealed with
bone wax, the wound was sutured, and rats were allowed to recover.
Physiological parameters, including mean arterial blood pressure,
blood gases, and glucose concentration, were measured during the
experiment. During the recovery period, rats were assessed for
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forelimb flexion and contralateral circling to confirm the procedures.
No cases of seizure were observed during the experiments. Rectal
temperature was maintained at 37⫾0.5°C with the use of a
thermistor-controlled heating blanket. Free access to food and water
was allowed after recovery from anesthesia. Rats were kept in
air-ventilated cages at 24⫾0.5°C for the duration of the experiment.
Morphometric Measurement of
Hemorrhage Volume
Rats were assigned to 2 experimental groups: a normoglycemic
group (control; n⫽8) and a hyperglycemic group treated with
streptozotocin (n⫽8). Three days after the injection of streptozoto-
cin, serum glucose level was obtained by tail vein sampling before
and after (measured daily for 2 days) the induction of ICH.
Seventy-two hours after the operation, rats were anesthetized by the
same methods and killed by decapitation. The brain was removed
immediately and fixed with 4% phosphate-buffered paraformalde-
hyde. Fixed brains were cut coronally through the needle entry site
(identifiable on the brain surface), and then serial slices (1 mm thick)
both anterior and posterior to the needle entry site were obtained.
Physiological Parameters
Normoglycemic Group
Baseline
pH 7.41⫾0.03
PaO2, mm Hg 109.1⫾10.2
PaCO2, mm Hg 38.3⫾1.0
Rectal temperature, °C 36.8⫾0.2
MABP, mm Hg 88⫾3
Values are means⫾SD. One-way analysis of variance revealed no significant intergroup difference
for any variable. ICH indicates intracerebral hemorrhage; MABP, mean arterial blood pressure.
Digital photographs of the serial slices were taken, and the size of
hemorrhage was measured with an image analyzer program (Image
Pro-Plus, Media Cybernetics). Total hematoma volume was calcu-
lated by summing the clot area in each section and multiplying by the
distance between sections.
Measuring Brain Water Contents
Rats were assigned to 2 experimental groups: one group consisting of
17 normoglycemic rats and the other group consisting of 17
hyperglycemic rats. Two days after operation, the brains were
harvested, and the cerebellum and the brain stem were removed and
then divided into 2 hemispheres along the midline. The wet weight
of the hemispheres was measured. The hemispheres were incubated
in an oven at 100°C for 24 hours to obtain the dry weight. Water
content was expressed as percentage of wet weight; the formula for
calculation was as follows: (wet weight⫺dry weight)/(wet
weight)⫻100.20
TUNEL Assay
Rats used for terminal deoxynucleotidyl transferase–mediated
dUTP-biotin nick end labeling (TUNEL) staining were killed with an
overdose of sodium pentobarbital at 72 hours after collagenase
injection (hyperglycemia: n⫽6; normoglycemia: n⫽6). The TUNEL
procedure for in situ detection of DNA fragmentation was per-
formed.21,22 In brief, air-dried sections were postfixed in 4% para-
formaldehyde for 15 minutes at room temperature. After they were
washed 3 times in Tris-HCl (pH 7.7), sections were treated with 2%
H2O2 for 10 minutes at room temperature to quench endogenous
peroxidase activity. Sections were then incubated with terminal
deoxynucleotidyl transferase enzyme solution containing 25 mmol/L
Tris-HCl (pH 6.6), 200 mmol/L potassium cacodylate, 0.25 mg/mL
bovine serum albumin, 2.5 mmol/L CoCl2, 40 ␮mol/L biotinylated-
16-dUTP, and 500 U/mL terminal deoxynucleotidyl transferase
(Boehringer-Mannheim) at 37°C for 1 hour. Sections were dipped in
300 mmol/L NaCl and 30 mmol/L sodium citrate for 15 minutes at
room temperature to terminate reactions. After sections were washed
3 times in Tris-HCl (pH 7.7) and subsequently blocked with PBS
(pH 7.4) containing 10% normal goat serum and 0.3% Triton X-100,
biotinylated-16-dUTP was visualized by the avidin-biotin method
with 0.05% 3,3⬘-diaminobenzidine tetrahydrochloride and 0.005%
H2O2. Cell density counts were performed on sections lightly
counterstained with toluidine blue stain. A single axial section
through the center of the hemorrhagic lesion was analyzed. Square
sampling regions (300⫻300 ␮m) were used for cell counting. Eight
sampling regions were placed along the periphery. The TUNEL-
positive cells were identified and counted. Total counts in these
sampling regions were converted into cell densities for quantification
and comparison between treatment groups.
Behavioral Testing
Behavioral testing was performed by 2 individuals blinded to the
assignment of rats (total n⫽28) before and 3 days after ICH with the
use of the modified limb placing test, which is a modified version of
a test previously described in the literature.23 The test consists of 2
limb-placing tasks that assess the sensorimotor integration of the
Hyperglycemic Group
After ICH Baseline After ICH
7.40⫾0.02 7.39⫾0.01 7.38⫾0.07
107.3⫾8.2 108.1⫾9.5 112.1⫾9.7
38.1⫾1.3 37.8⫾0.9 38.4⫾1.2
36.8⫾0.2 36.6⫾0.3 36.7⫾0.1
85⫾3 86⫾3 87⫾2
 Song et al Aggravation of Brain Edema in ICH by Hyperglycemia 2217

Figure 2. Hematoma volume (a) and

example of brain slices (b) 72 hours after
intracerebral infusion of collagenase. Val-
ues are mean⫾SD. Hematoma volumes
were not different between groups.
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forelimb and the hind limb by checking responses to tactile and Size of Hemorrhage
proprioceptive stimulation. First, the rat is suspended 10 cm over a Rats infused with 0.23 U of collagenase developed large
table, and the stretch of the forelimbs toward the table is observed hemorrhages in the striatum that were evident by 24 hours
and evaluated as follows: normal stretch, 0 points; abnormal flexion, (Figure 2). The size of hemorrhage was 8.99⫾4.51 mm3
1 point. Next the rat is positioned along the edge of the table, and its
forelimbs are suspended over the edge of the table and allowed to
(n⫽8) in the hyperglycemic group and 8.87⫾3.61 mm3
move freely. Each limb (forelimb, second task; hind limb, third task) (n⫽8) in the normoglycemic group (Figure 2) by 72 hours.
is pulled down gently, and retrieval and placement are checked. There was no significant difference between the sizes of
Finally, the rat is placed toward the table edge to check for lateral
placement of the forelimb. The 3 tasks are scored in the following
manner: normal performance, 0 point; performance with a delay (2
seconds) and/or incomplete, 1 point; no performance, 2 points. Seven
points indicates maximal neurological deficit, and 0 points indicates
normal performance.

Statistical Analysis
All data in this study are presented as mean⫾SD. Data from different
animal groups were analyzed by the Mann-Whitney U test and
unpaired Student t test. The differences were considered significant
at probability values ⬍0.05.

Results
Physiological Parameters
All animals survived the experiments. The serum glucose
level of the hyperglycemic group (n⫽31) was 394.0⫾180.3
mg/dL, and that of normoglycemic group (n⫽31) was
97.5⫾27.4 mg/dL (P⬍0.01, unpaired t test; Figure 1). Other
physiological parameters, including mean arterial blood pres-
sure, blood gases, and body temperatures, were not signifi- Figure 3. Brain water content 48 hours after intracerebral infu-
sion of collagenase. Hyperglycemia increased brain water con-
cantly different among experimental groups before, during, or tent in lesioned and contralateral hemispheres. Values are
after ICH (Table; unpaired t test). mean⫾SD. *P⬍0.01 compared with normoglycemic group.
 2218 Stroke September 2003

hemorrhage between groups (P⫽1.00, Mann-Whitney U
test).
Minor intraventricular hemorrhage (IVH) occurred in a
few rats (n⫽3 of 14, normoglycemic group; n⫽3 of 14,
hyperglycemic group), which did not significantly affect the
behavioral test results measured at 3 days after ICH induction
(modified limb placing test score: 6.4⫾0.2 in ICH⫹IVH
group, n⫽6; 6.3⫾0.4 in ICH only group, n⫽22; P⫽0.95,
Mann-Whitney U test).
Brain Edema Formation
Brain water content of the lesioned (left) hemisphere was
81.0⫾0.5% in the hyperglycemic group (n⫽17) and
78.9⫾0.8% in the normoglycemic group (n⫽17) (P⬍0.01,
unpaired t test); brain water content of the nonlesioned (right)
hemisphere was 78.7⫾0.6% in the hyperglycemic group and
77.3⫾1.1% in the normoglycemic group (P⬍0.01, unpaired t
test; Figure 3). Brain water content of hyperglycemic rats
increased in both lesioned and nonlesioned hemispheres
compared with the normoglycemic group.
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TUNEL-Positive Cell Density After ICH
TUNEL staining revealed a high density of positive cells
within the hemorrhage lesion itself as well as in the surround-
ing periphery (Figure 4). Brain sections that were counter-
stained with toluidine blue showed that the TUNEL-positive
cells were not likely to be infiltrating leukocytes (Figure 4).
Quantitative analysis showed regional TUNEL-positive cell
differences between the groups (n⫽6). The hyperglycemic
group (923.1⫾74.3 cells per square millimeter) showed
significantly more TUNEL-positive cells than the normogly-
cemic group (301.7⫾91.5 cells per square millimeter;
P⬍0.01, unpaired t test).
Discussion
Using a rat model of ICH, we have shown that hyperglycemia
may cause more profound brain edema. Numbers of TUNEL-
positive cells were significantly increased in rats with hyper-
glycemia. The size of hemorrhage and physiological param-
eters (except for serum glucose level) were not significantly
different between normoglycemic and hyperglycemic rats.

Figure 4. Results of TUNEL staining. a, b, Representative section in the perihematomal zone of the hemorrhagic lesion shows the pres-
ence of TUNEL-positive stained cells (a, hyperglycemic group; b, normoglycemic group). Counterstaining with toluidine blue indicates
that polymorphonuclear leukocytes do not show TUNEL-positive staining. Note the more abundant TUNEL-positive cells in the hyper-
glycemic group (a). c, Density of TUNEL-positive stained cells per square millimeter. Values are mean⫾SD; n⫽6 per group. *P⬍0.01
compared with normoglycemic group.
 Song et al Aggravation of Brain Edema in ICH by Hyperglycemia
In our study brain edema after ICH with hyperglycemia
increased in both lesioned and nonlesioned hemispheres, and
this may be more attributable to vasogenic edema. Brain
edema is generally divided into cytotoxic and vasogenic
types, depending on whether the BBB is intact or disrupt-
ed.20,24 –27 The brain edema of experimental ICH is regarded
as a combination of cytotoxic and vasogenic edema. Brain
edema forms as a result of BBB disruption and the local
generation of osmotically active substances. Brain edema was
increased not only in the perihematomal region but also in the
more distant structures, even in contralateral basal ganglia,
possibly because of increased BBB permeability and diaschi-
sis, in rat ICH models20 and human subjects with ICH.28
Our results revealed that hyperglycemia may aggravate
brain edema, culminating in neural cell death around the
hematoma, with an increase in TUNEL-positive cells. Colla-
genase itself, used in this study for the induction of ICH, did
not cause cell death in vitro (10 to 15 U /mL).22 Apoptosis is
reported to be involved in the pathophysiology of cell death
after ICH.22 However, the triggering events that are respon-
sible for initiating the apoptotic cascade after ICH remain to
be defined. The activated blood components (eg, thrombin,
iron, and heme) may induce high levels of cytokine activi-
ty.22,29 Inflammatory cytokines such as tumor necrosis
factor-␣ (TNF-␣) and interleukin-1 (IL-1) may be involved in
hyperglycemia-induced brain edema. Administration of IL-1
and TNF-␣ induces opening of the BBB and causes vaso-
genic brain edema.30 –32 In vitro studies demonstrated that
hyperglycemia increases the secretion of IL-1␤ in cultured
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human aortic endothelial cells.33 Mouse uterine epithelial
cells cultured in high glucose showed an increase in TNF-␣
production via an increase in IL-1␤ secretion.34 Acute hyper-
glycemia increases the level of circulating TNF-␣ in humans;
this effect is more pronounced in patients with impaired
glucose tolerance.35
Previous experimental studies have demonstrated that hy-
perglycemia worsens acute BBB injury after transient fore-
brain ischemia in rats and accentuates brain edema.5,6,36,37
The summarized speculations for hyperglycemia-induced
brain injury are as follows. (1) Free radical formation is
increased with hyperglycemia-induced brain injury. The free
radicals and nitric oxide increase BBB permeability and brain
edema.38 – 40 (2) Hyperglycemia induces bradykinin-mediated
brain edema with inflammation. Bradykinin is a potent
vasodilator and induces brain edema in animal models41;
bradykinin B2 receptors play a role in the formation of
vasogenic brain edema in a cold injury model in rats, and B2
receptor antagonist reduces vasogenic edema.42,43 Bradykinin
increases BBB permeability and facilitates extravasation by
dilation of arterial blood vessels.43 (3) Hyperglycemia exerts
its effect on cell death by inducing inflammatory reactions in
ICH.44,45 (4) Hyperglycemia causes acute elevation of cyto-
solic free calcium as a result of increased calcium influx.46,47
Rising calcium concentration in the cytoplasm causes cal-
cium influx into mitochondria and disrupts mitochondrial
membrane potential and normal metabolism, leading to cell
death. The calcium channel blocker (S)-emopamil reduces
brain edema from collagenase-induced hemorrhage in rats,
possibly by reducing calcium entry and sodium exchange.48
2219
In summary, our data provide initial evidence that hyper-
glycemia may aggravate brain edema with neural cell death in
an experimental rat ICH model.
Acknowledgments
This work was supported by grant 02-1996-3280 from the Seoul
National University Hospital Research Fund. We thank Sung Chu
and Yonjae Chung-Chu for preparing the figures and for editorial
support.
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