HMG-CoA Reductase Inhibitor, Atorvastatin, Promotes Sensorimotor Recovery,
Suppressing Acute Inflammatory Reaction After Experimental Intracerebral Hemorrhage
Keun-Hwa Jung, Kon Chu, Sang-Wuk Jeong, So-Young Han, Soon-Tae Lee, Jin-Young Kim,
Manho Kim and Jae-Kyu Roh
Stroke. 2004;35:1744-1749; originally published online May 27, 2004;
doi: 10.1161/01.STR.0000131270.45822.85
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HMG-CoA Reductase Inhibitor, Atorvastatin, Promotes
Sensorimotor Recovery, Suppressing Acute Inflammatory
Reaction After Experimental Intracerebral Hemorrhage
Keun-Hwa Jung, MD; Kon Chu, MD; Sang-Wuk Jeong, MD; So-Young Han, MS; Soon-Tae Lee, MD;
Jin-Young Kim, BS; Manho Kim, MD, PhD; Jae-Kyu Roh, MD, PhD
Background and Purpose—Statins have neuroprotective effects on ischemic stroke. They modify the endothelial function,
increase blood flow, and inhibit thrombus formation, which are independent of lipid-lowering effects. However, whether
statins have a protective effect toward hemorrhagic stroke is yet unknown. To test this possibility, we attempted to
determine the effect of atorvastatin on experimental intracerebral hemorrhage (ICH).
Methods—ICH was induced using stereotaxic infusion of collagenase into the left basal ganglia in adult rats. Atorvastatin
(1 mg/kg or 10 mg/kg) or phosphate-buffered saline was administered for 2 weeks. To monitor the sensorimotor deficits,
limb placing and Rotorod tests were performed. Hematoma volume, brain water content, and hemispheric atrophy were
analyzed. Immunohistochemical staining for myeloperoxidase (MPO), microglia (OX42), inducible nitric oxide
synthase (iNOS), or endothelial nitric oxide synthase (eNOS) was performed. Perihematomal cell death was determined
by TUNEL staining.
Results—The atorvastatin-treated ICH group showed better performance on Rotorod and limb placing tests when compared
with the vehicle-treated group (P⬍0.01). The hematoma volumes between groups were not different, but the brain water
content and hemispheric atrophy were reduced in the atorvastatin-treated ICH group. Atorvastatin reduced TUNELpositive cells, iNOS expression, and MPO-positive or OX42-positive cells in the perihematomal regions in a
dose-dependent manner, whereas it increased eNOS expression.
Conclusion—The present study shows that atorvastatin reduces the perihematomal cell death via antiinflammation, which
is associated with sensorimotor recovery after experimental ICH. (Stroke. 2004;35:1744-1749.)
Key Words: HMG-CoA reductase inhibitors 䡲 intracerebral hemorrhage 䡲 brain edema
䡲 apoptosis 䡲 inflammation
I
ntracerebral hemorrhage (ICH) represents 15% of all
strokes in Western populations and a higher proportion
(20% to 30%) in Asian and black populations.1 The mechanisms by which cellular injury occurs after ICH include
mechanical destruction or displacement caused by blood
accumulation.2 Inflammation and impairment of cerebral
blood flow (CBF) around the blood clot contribute to delayed
neuronal death.3,4 The suppression of the inflammatory response has been reported to reduce brain edema and tissue
damage and improve functional outcome in experimental
ICH.5– 8
Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMGCoA) reductase inhibitors, have been shown to have many
pleiotropic properties on the nervous and vascular systems,
independent of cholesterol-lowering effect, and provide neuroprotection with beneficial effects on endothelial function,
immunomodulation, and impeding excitotoxic cell injury via
antiinflammatory actions and angiogenesis/neurogenesis.9 –22
The putative neuroprotective actions of statins may lead to
functional restoration after ICH. However, the effects of
statins in the ICH paradigm are not known at present. In the
present study, we investigate whether atorvastatin, when
administered after ICH, can reduce edema formation, inflammation, and cell death, and subsequently promote neurological recovery in a rat model of ICH.
Materials and Methods
Induction of Intracerebral Hemorrhage and
Atorvastatin Administration
Male Sprague-Dawley rats (12 weeks old; Genomics, Seoul, ROK),
weighing 200 to 220 grams, were used in this experiment and our
protocols were institutionally approved in accordance with NIH
Received January 23, 2004; final revision received March 10, 2004; accepted March 29, 2004.
From Stroke and Neural Stem Cell Laboratory in Clinical Research Institute (K.-H.J., K.C., S.-Y.H., S.-T.L., J.-Y.K., M.K., J.-K.R.), Department of
Neurology, Seoul National University Hospital, Seoul National University, Seoul, South Korea; the Department of Neurology (K.C.), Seoul National
Hospital, Seoul, South Korea; and the Department of Neurology (S.-W.J.), Ilsan Paik Hospital, Inje University, Goyang, South Korea.
K.-H.J. and K.C. contributed equally to this work.
Correspondence to Dr Jae-Kyu Roh, Department of Neurology, Seoul National University Hospital, 28, Yongon-Dong, Chongro-Gu, Seoul, 110-744,
South Korea. E-mail rohjk@snu.ac.kr
© 2004 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org
DOI: 10.1161/01.STR.0000131270.45822.85
1744
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by guest on February 23, 2013
Jung et al
Guide for the Care and Use of Laboratory Animals. Experimental
groups consisted of rats subjected to ICH (n⫽99) and normal control
rats (n⫽27). Experimental ICH was induced by the stereotaxic
intrastriatal administration of bacterial collagenase type IV (Sigma),
as previously described.23,24 After an intraperitoneal injection of 1%
ketamine (30 mg/kg; Sigma) and xylene 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 through a burr hole 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). ICH was induced by the
administration of 1 ␮L containing 0.23 U of collagenase over 5
minutes. After infusion, craniotomy holes were sealed with bone wax
and wounds were sutured. Rectal temperature was maintained at
37⫾0.5°C using a thermistor-controlled heating blanket.
Six hours after ICH induction (considering the recovery time from
anesthesia), rats were fed atorvastatin (prescription formulation;
Pfizer Inc), using 20-mm feeding needles (Popper and Sons Inc),
dissolved in phosphate-buffered saline, 1 mg/kg (n⫽21) or 10 mg/kg
(n⫽39) daily for 3 days or 14 days (to the rats assigned for the
behavioral testing). ICH rats fed vehicle alone served as ICH control
group (n⫽39), and normal rats fed vehicle alone (n⫽18) or 10 mg/kg
atorvastatin (n⫽9) served as normal control group.
Behavioral Testing
Behavioral testing was performed weekly for atorvastatin-treated (10
mg/kg) or vehicle-treated ICH group (n⫽9 per group) using the
modified limb placing (MLPT) and Rotorod tests,23 which were
monitored by blinded investigators for group allocation.
Morphometric Measurement of Hematoma
Volume and Hemispheric Atrophy
At 3 days after ICH, brains from atorvastatin-treated (10 mg/kg) or
vehicle-treated ICH rats (n⫽9 per group) were cut coronally through
the needle entry site (identifiable on the brain surface), and then
serial slices (1-mm thickness) both anterior and posterior to the
needle entry site were obtained. Digital photography of the serial
slices was taken and hematoma volume was measured using image
analyzer program (Image Pro-Plus; Media Cybernetics). The total
hematoma volume (mm3) was calculated by summing the clot area in
each section and multiplying by the distance between sections.24 At
42 days after ICH, the rats used for behavior test were euthanized to
analyze hemispheric atrophy. Three sections through the needle
entry site and sites 1.0 mm anterior and 1.0 mm posterior to plane
were Nissl-stained. The morphometric analyses involved the area of
the striatum, cerebral cortex, and ventricle, as well as the whole
hemisphere. The total hemispheric areas of each section were traced
and measured using image analyzer. The hemispheric atrophy was
expressed as a percentage of contralateral hemispheric area.23
Analysis of Brain Water Content, Histology,
and Immunohistochemistry
We determined the endpoint of 3 days for experiments on water
content, histology, and immunohistochemistry, because inflammatory reaction has been shown to be maximal at 48 to 72 hours after
ICH.3 Atorvastatin-treated (1 mg/kg or 10 mg/kg) or vehicle-treated
ICH rats and atorvastatin-treated (10 mg/kg) or vehicle-treated
normal rats (n⫽9 per group) were euthanized to measure brain water
content. The brains were divided into 2 hemispheres along the
midline, and the cerebellum and the brain stem were removed. The
brain samples were immediately weighed on an electronic analytical
balance to obtain the wet weight. The samples were then dried in a
gravity oven at 100°C for 24 hours to obtain the dry weight. Water
content was expressed as a percentage of wet weight: the formula for
calculation was (wet weight⫺dry weight)/(wet weight) ⫻ 100.24
At the same time, atorvastatin-treated (1 mg/kg or 10 mg/kg) or
vehicle-treated ICH rats (n⫽9 per group) and vehicle-treated normal
rats (n⫽6) were euthanized for histological analysis. We removed
the brains and cryopreserved tissue blocks for cryostat sectioning
(30 ␮m thickness). Euthanization or immunohistochemistry was
Effect of Atorvastatin on ICH
1745
processed as described previously.23,24 Rabbit anti-human myeloperoxidase (MPO) (1:100; DAKO Corporation) and mouse anti-rat
OX42 primary antibodies (1:500; Chemicon) were used as markers
for neutrophil and microglia, respectively. MPO-positive or OX42positive cells were identified and counted. The iNOS and eNOS
immunohistochemistries were performed with anti-iNOS antibody
(1:300, mouse monoclonal; Transduction Laboratories) and antieNOS antibody (1:500, rabbit polyclonal; Transduction Laboratories), respectively. FITC-conjugated anti-mouse IgG (1:100; Jackson
ImmunoResearch) and Cy3-conjugated anti-rabbit IgG antibodies
(1:500; Jackson ImmunoResearch) were used for the secondary
antibodies.
Western Blot
Western blotting was performed on perihematomal tissues dissected
out from a 2-mm-thick coronal brain slice cut at the level of the
needle insertion. A similar segment was isolated in normal control
rats. Western blotting of eNOS (n⫽3 per group) was performed with
a rabbit polyclonal eNOS antibody (1:500; Transduction Laboratories) as previously described.25 Using the same membrane, we also
performed immunoblotting of ␤-actin with a mouse anti–␤-actin
monoclonal antibody (1:2000; Sigma) to standardize the expression
of eNOS. Equal amounts of protein (20 ␮g) for each group were
assayed and immunoreactivity was visualized by enhanced
chemiluminescence.
TUNEL Assay
Atorvastatin-treated (1 mg/kg or 10 mg/kg) or vehicle-treated ICH
rats (n⫽9 per group) and vehicle-treated normal rats (n⫽6) were
euthanized with sodium pentobarbital at 3 days after ICH for
TUNEL staining. The TUNEL procedure for in situ detection of
DNA fragmentation was performed as previously described.24,26
Only TUNEL-stained cells that showed deep punctate staining with
perinuclear chromatin condensation were considered apoptotic and
were included for quantitation. Cell density counts were performed
on sections counterstained with toluidine blue.
Cell Quantification
Quantitative analysis of the positively stained cell number was
performed in the perihematomal regions of 3 axial sections (1-mm
width) staining through the center of the hemorrhagic lesion by 2
independent investigators who were blinded to the group allocation.
Total counts in the measured sections were converted into cell
densities for comparison between the ICH groups.
Statistical Analysis
All data in this study are presented as the mean⫾SD. Data were
analyzed by unpaired Student t test if they were normally distributed
(Kolmogorov-Smirov test, P⬎0.05). Otherwise, we used Mann–
Whitney U test and specified the test used. Two-tailed value of
P⬍0.05 was considered significant.
Results
Physiological Parameters
All animals survived the surgery. The physiological parameters, including mean arterial blood pressure, blood
gases, serum glucose, and body temperatures, were not
significantly different in any experimental groups before,
during, or after ICH.
Atorvastatin Improved the Sensorimotor Deficits
After Experimental Rats
The atorvastatin-treated (10 mg/kg) ICH group showed a
better performance on the MLPT after 21 days and the
Rotorod after 14 days compared with the vehicle-treated
group, and the effects persisted up to 28 days. At day 28, the
Rotorod performance showed 80% improvement of pre-
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1746
Stroke
July 2004
Figure 1. Modified limb placing test and
Rotorod test. Atorvastatin-treated ICH
group showed a better performance on
the MLPT (A) and the Rotorod (B) compared with the vehicle-treated group;.
n⫽9 per group; *P⬍0.05 compared with
ICH-only group.
trained level, whereas the vehicle-treated ICH group showed
40% (Figure 1; n⫽9 for each group, P⬍0.05, Mann–Whitney
U test). The initial body weights and those over the course of
4 weeks were similar.
Atorvastatin Reduced Brain Water Content and
Long-Term Hemispheric Atrophy
The hematoma volume was 19.16⫾1.99 mm3 in the vehicletreated ICH group and 18.83⫾1.58 mm3 in the atorvastatintreated group by 3 days (Figure 2A; n⫽9 for each group,
P⫽0.66, Student t test). Brain water contents of the lesioned
hemisphere were reduced in atorvastatin-treated ICH group
with dose-dependency (Figure 2B; n⫽9 for each group,
P⬍0.05, Mann–Whitney U test). Brain water contents of the
nonlesioned hemisphere were not different. Hemispheric area
analysis showed an atrophy of lesioned hemisphere in both
vehicle-treated and atorvastatin-treated ICH groups (Figure
2C; n⫽9 for each group). However, the atorvastatin-treated
ICH group showed lesser hemispheric and striatal atrophy
than the vehicle-treated group (15.00⫾2.16% versus
24.52⫾4.79% and 15.50⫾2.38% versus 27.00⫾3.91%, respectively; P⬍0.05, Mann–Whitney U test), whereas cortical
atrophy was not different between both groups (5.75⫾2.06%
versus 6.75⫾2.50%; P⫽0.46).
Atorvastatin Decreased TUNEL-Positive Cells in
the Perihematomal Regions
After 3 days, a high density of TUNEL-positive stained cells
was present within the hemorrhage as well as in the surrounding periphery (Figure 3). Counterstaining with toluidine blue
revealed that the TUNEL-positive cells were not leukocytes.
TUNEL-positive cells in atorvastatin-treated groups were
significantly decreased by 42% (10 mg/kg) and 24% (1
mg/kg) compared with vehicle-treated group (n⫽9 for each
group, P⬍0.05, Student t test).
Figure 2. Hematoma volume, brain water
content, and hemispheric atrophy.
Hematoma volume was not different
between vehicle-treated and
atorvastatin-treated ICH groups (A). Brain
water content was reduced in the
lesioned hemisphere (B), and hemispheric and striatal atrophy of lesioned
side were reduced in the atorvastatintreated ICH group (C); n⫽9 per group for
each experiment; bars represent
mean⫾SD; *P⬍0.05 compared with
vehicle-treated group.
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Jung et al
Effect of Atorvastatin on ICH
1747
Figure 3. Perihematomal cell death.
More abundant TUNEL-positive cells
were observed in the perihematomal
region of vehicle-treated ICH group (A)
than atorvastatin-treated group (B).
Quantitative analysis showed less
TUNEL-positive cells in the
atorvastatin-treated ICH group in a
dose-dependent manner (C); n⫽9 per
group; scale bar⫽50 ␮m. Bars represent mean⫾SD. *P⬍0.05 compared
with ICH-only group.
Atorvastatin Decreased iNOS Expression and
MPO-Positive or OX42-Positive Cells
Atorvastatin Increased eNOS Expression in the
Perihematomal Regions
iNOS immunoreactivity was increased in the perihematomal
regions, but it was attenuated by ⬇50% in the atorvastatintreated (10 mg/kg) ICH group (Figure 4A and 4B). A
significant infiltration of MPO-positive or OX42-positive
cells adjacent to the hematoma was also detected 3 days after
ICH (Figure 4C to 4F). Quantitative analysis (Figure 4G and
4H) showed 36% reduction in MPO⫹ and 42% in OX42⫹
cells in the atorvastatin-treated (10 mg/kg) ICH group compared with the vehicle-treated group, whereas it showed a
minimal reduction in the atorvastatin-treated (1 mg/kg) ICH
group.
The eNOS expression in the perihematomal regions was increased in the atorvastatin-treated (10 mg/kg) ICH group compared with the vehicle-treated group (Figure 5). In addition, a
2-fold increase in eNOS protein was detected by Western
blotting after 3 days of 10 mg/kg treatment, whereas the increase
was minimal after 1 mg/kg treatment (Figure 5C).
Discussion
In the present study, atorvastatin, when administered to
collagenase-induced ICH model, reduced hemispheric water content and perihematomal cell death, decreased iNOS
Figure 4. Immunohistochemistry for
iNOS, MPO, and OX42 in the perihematomal region. Immunohistochemistry
for iNOS in vehicle-treated ICH rats
showed an increment of iNOS expression around the blood clot (A), whereas
this increment was attenuated in
atorvastatin-treated ICH rats (B). MPO⫹
neutrophils (C, D) and OX42⫹ (E, F)
microglias were clustered around the
blood clot. The numbers of MPO⫹ and
OX42⫹ cells were decreased by atorvastatin treatment. Quantitative analysis
(G, H) shows a dose-dependent reduction in MPO⫹ and in OX42⫹ cells; n⫽9
per group; magnification ⫻100; bars
represent mean⫾SD; *P⬍0.05 compared with vehicle-treated ICH group.
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Stroke
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Figure 5. Immunohistochemistry and Western blotting for
eNOS expression in the ICH brain. Atorvastatin-treated (10
mg/kg) ICH rats (B) show the increased eNOS expression in
the perihematomal regions compared with vehicle-treated
rats (A). Arrows indicate the increased expression of eNOS in
the vessel. Magnification ⫻200. Western blot of eNOS in the
ICH brain shows a dose-dependent eNOS expression by
atorvastatin treatment. The bands of ␤-actin in the lower
panel are shown for internal standards. n indicates normal
control; v, vehicle; 1, 1 mg/kg atorvastatin; 10, 10 mg/kg
atorvastatin.
expression, decreased infiltration of leukocytes and microglia, and increased eNOS expression in the perihematomal
regions while concomitantly improving the functional
outcome and decreasing the long-term hemispheric atrophy. Our data provide initial evidence that statin exerts
protection against ICH via modulation of inflammation.
Despite the similar hematoma volumes between groups,
the neurological deficit after ICH was significantly ameliorated by atorvastatin. These findings suggest that sensorimotor deficits after ICH are not simply attributed to the
mechanical destruction by the hematoma, supporting that
inflammatory response, brain edema, or impairment of
CBF around the blood clot might also contribute to
functional impairment.2,3
Cell necrosis, perihematomal cell apoptosis, and axonal
loss lead to brain atrophy after ICH, which progresses up
to 3 months.2– 4 In the present study, atorvastatin treatment
for 14 days (targeting a period of active inflammation or
cell death after ICH) reduced a brain atrophy when
measured at 42 days, and this was associated with attenuation of apoptosis. Apoptosis after ICH begins at 24 hours,
peaks at 3 days, and continues at least 4 weeks.27 The
perihematomal ischemia and inflammatory responses have
been suggested to mediate the apoptosis.3,27 Therefore, the
modulation of inflammation and preservation of CBF by
atorvastatin might help ameliorate the cell death after ICH,
giving the reduction in hemispheric atrophy and the
functional improvement. However, it remains to be established if the injection of bacterial collagenase by itself
causes an inflammatory response and worsened accompanying cerebral edema. Further studies using autologous
blood-injected ICH models are needed.
Toxic blood components can induce an inflammatory
response in and around the hematoma.4 Infiltrating inflammatory cells or activated microglia enhance the production
of proinflammatory cytokines, cyclooxygenase-2, and
iNOS, presumably inducing the signal pathway to mediate
the apoptosis.27,28 Inhibition of these inflammatory substances has been reported to reduce a brain edema and
tissue damage and improve functional outcome in experimental ICH.5– 8 Statins cause a dose-dependent inhibition
of multiple steps of leukocyte recruitment and migration
into the central nervous system, either directly by suppressing cell surface receptors and adhesion molecules on
nervous or vascular tissues or indirectly by binding to
leukocyte function-associated antigen.29 –31 Statins can also
suppress the proinflammatory mediators such as TNF-␣,
IFN-␥, and iNOS.9,32 Recent data have demonstrated that
statins act beneficially in numerous inflammatory conditions
such as cardiac transplantation (in humans)18 and experimental
autoimmune encephalomyelitis (in mice).20 –22 In our experiment, attenuation of inflammatory response by atorvastatin
might mediate decreasing brain edema and cell death.
Statins have also been shown to enhance CBF in
experimental stroke.14 –16 Statins upregulate eNOS in ischemic stroke12–15 and experimental subarachnoid hemorrhage.11 Enhancement of CBF and neuroprotective effects
of statins are completely absent in eNOS-deficient mice,15
indicating that enhanced eNOS activity by statins is the
predominant mechanism by which these agents protect
against cerebral injury. In our experiment, eNOS was
upregulated in the perihematomal regions. Thus, atorvastatin might attenuate the ischemic neuronal injury in the
perihematomal ischemic penumbra by enhancing CBF. To
clarify other pleiotropic effects of statin including neurogenesis and angiogenesis, further investigations are
needed.
The patients with ischemic stroke often undergo hemorrhagic stroke. Up to 30% of all ischemic strokes develop
hemorrhagic transformations, and thrombolytic therapy of
ischemic stroke can lead to serious complications of ICH.
Therefore, in practice, it is important to identify agents that
are safe and efficacious in treating both ischemic and
hemorrhagic stroke. Taken together with the pleiotropic
effects of statin in acute ischemic stroke, statin might be a
useful agent in these complex conditions.
Acknowledgments
This study was supported by the 21st Century Frontier Research
Fund of Ministry of Science & Technology (M102KL01000102K1201-01310), Republic of Korea.
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