Download Reduction and Restoration of Mitochondrial DNA Content

Reduction and Restoration of Mitochondrial DNA Content
After Focal Cerebral Ischemia/Reperfusion
Hong Chen, PhD; Chaur-Jong Hu, MD; Yong Y. He, MD; Ding-I Yang, PhD;
Jan Xu, PhD; Chung Y. Hsu, MD, PhD
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Background and Purpose—Oxidative damage of mitochondrial DNA (mtDNA) in the ischemic brain is expected after
ischemia/reperfusion injury. A recent study demonstrated limited patterns of mtDNA deletion in the brain after
ischemia/reperfusion. We studied the ischemia/reperfusion-induced global changes of mtDNA integrity and its
restoration in a rat model of transient focal ischemia in vivo.
Methods—Changes in mtDNA content in the ischemic brain were assessed with the use of a rat stroke model featuring
transient severe ischemia confined to the cerebral cortex of the right middle cerebral artery territory for 30 or 90 minutes.
A new long polymerase chain reaction method, using mouse DNA as an internal standard, was applied to measure the
relative content of intact rat mtDNA. Southern hybridization following alkaline gel electrophoresis was conducted in a
parallel study to confirm long polymerase chain reaction results.
Results—A reduction in mtDNA content was found after ischemia for 30 and 90 minutes. The mtDNA was restored to near
nonischemic levels 24 hours after 30- but not 90-minute ischemia.
Conclusions—These results confirm that ischemia/reperfusion causes mtDNA damages. Restoration of the mtDNA
content to nonischemic levels after 30-minute ischemia raises the possibility that mtDNA repair or repletion occurs after
brief ischemia. (Stroke. 2001;32:2382-2387.)
Key Words: cerebral ischemia, focal 䡲 DNA damage 䡲 DNA, mitochondrial 䡲 DNA repair 䡲 rats
M
itochondria play a central role in necrotic and apoptotic
cell death.1 A growing body of evidence has suggested
that defects in mitochondrial DNA (mtDNA), such as mutations or deletions, may underlie the molecular mechanisms of
various human diseases.2 In part because of the spatial
proximity of mtDNA to the electron transport system, where
oxidative phosphorylation occurs with the concomitant generation of free radicals or reactive oxygen species (ROS), the
probability of oxidative damage to mtDNA is several times
higher than nuclear DNA.3 Compelling evidence suggests
that ischemia/reperfusion induces mitochondrial dysfunction
by enhancing ROS generation, leading to the damage of
intracellular proteins, lipids, and DNA.4 Previous studies
have revealed specific patterns of mtDNA deletions, such as
3726, 4236, or 3867 bp, in transient global ischemia, traumatic brain injury, and focal ischemia.5,6 While the pathological consequence of such damages in brain injury models
remains unclear, a strong correlation between the accumulation of oxidative mtDNA damage and the aging process has
been established.7 Several cellular mechanisms, including
endogenous free radical scavengers and intracellular antioxidants, are available to prevent mtDNA damage by ROS. In
addition, the repair machinery removes the oxidative lesions
on mtDNA. Furthermore, the replication systems may replenish the lost mtDNA. Both the repair and replication systems
of mtDNA are distinct from those of nuclear DNA. For
example, mitochondria appear to lack a nucleotide excision
repair mechanism.8 Unlike nuclear DNA, mtDNA replication
is coupled with the transcription process that is independent
of the progression of cell cycle or replication of nuclear
DNA.9 Nuclear DNA damages and subsequent repair occur
after forebrain ischemia/reperfusion injury.10 –12 Limited patterns of mtDNA deletions have also been demonstrated in
transient focal ischemia.6 However, the extent of mtDNA
damage and its repair and/or replication have not been
previously assessed in animal stroke models.
Several approaches have been reported to measure the
extent of mtDNA damage and repair. However, most of
these methods allow the detection of a few deletion
patterns. Quantitative assessment of the extent of mtDNA
damage is limited by a number of technical pitfalls. These
include variations in the yield of mtDNA during its
isolation and the extent of mtDNA damage in the preparation process.8 We use a new long polymerase chain
reaction (LPCR) method to explore changes in mtDNA
content after ischemia/reperfusion in a rat stroke model,
Received March 22, 2001; final revision received June 16, 2001;accepted June 22, 2001.
From the Center for the Study of Nervous System Injury and Department of Neurology, Washington University School of Medicine, St Louis, Mo.
Drs Chen and Hu contributed equally to this study.
Correspondence to Chung Y. Hsu, MD, PhD, Center for the Study of Nervous System Injury and Department of Neurology, Washington University
School of Medicine, 660 S Euclid Ave, Campus Box 8111, St Louis, MO 63110. E-mail [email protected]
© 2001 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org
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Chen et al
applying a novel internal standard to minimize the variations inherent to the assay procedures.
Materials and Methods
Focal Cerebral Ischemia/Reperfusion in Rats
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A total of 48 male Long-Evans rats (weight, 300 to 350 g) were used
in the present study. All animal procedures were approved by the
Animal Care Committee of Washington University Medical Center
and followed the guidelines for animal care established by the
National Institutes of Health. The surgical procedures for focal
ischemia affecting the cerebral cortex in the middle cerebral artery
(MCA) territory have been previously described in detail13–15 with
modifications. Briefly, the animals were anesthetized with a single
injection of chloral hydrate (400 mg/kg IP). Body temperature was
maintained at 37⫾0.5°C with a servo-controlled lamp connected to
a rectal thermometer throughout surgery until recovery from anesthesia (return of righting reflexes). Microsurgical techniques were
used to expose the right MCA. The MCA was ligated at the level of
the inferior cerebral vein with 10-0 ophthalmic suture. Both common
carotid arteries were then exposed by a midline cervical incision and
clamped with vascular clips. At the end of ischemia (30 or 90
minutes), the suture was removed from the MCA, and common
carotid arteries were unclamped to allow reperfusion for various
period of time (0, 0.5, 4, or 24 hours). The animals were then
decapitated under anesthesia to collect the brains. The right (ischemic) and left (control) MCA cortices were immediately dissected and
separated from the subcortical structures.13 Samples were snapfrozen in liquid nitrogen and stored at ⫺70°C until use. In this
model, ischemia for 30 minutes results in little or no infarction in the
right MCA territory 24 hours after reperfusion. Ischemia for 90
minutes consistently generates a large infarct confined to the right
MCA cortex at 24 hours.14,16
Total DNA Isolation
Total DNA was purified with the use of the genomic DNA isolation
kit from Qiagen. The frozen tissue was homogenized with a
homogenizer and lysed with G2 buffer supplied in the kit. The
amount of DNA was quantitatively determined with the use of
PicoGreen (Molecular Probes), as previously described.17,18
LPCR for Quantification of mtDNA
The mtDNA derived from rat or mouse brains was first linearized by
digesting with restriction enzyme SacII (Promega). The quantification of intact mtDNA was achieved by means of a LPCR method19,20
with modifications. The reaction mixtures contained 0.4 ng of rat
total DNA, 4 pmol of each oligonucleotide primer, 400 ␮mol/L
dNTP mixtures, and 0.5 U of LA Taq enzyme (Takara Shuzo Co,
Ltd) in a total volume of 10 ␮L. The same amount (0.4 ng) of total
DNA derived from mouse brains was added to the polymerase chain
reaction (PCR) reaction mixture to serve as an internal standard. The
primers for the amplification of 14.3-kb mitochondrial genomes of
both rat and mouse were 5⬘-ATATTT-ATCACTGCTGAGTCCCGTGG-3⬘ (forward) and 5⬘-AATTTCGGTTGGGGTGACCTCGGAG-3⬘ (reverse). The conditions for PCR consisted of denaturation for 1 minute at 94°C followed by 26 cycles of
denaturation at 94°C for 15 seconds, annealing and extension at 68°C
for 10 minutes, and a final extension at 72°C for 10 minutes. The
PCR products were digested with restriction enzyme NcoI (Promega)
at 37°C for 2 hours and fractionated through a 1% agarose gel
containing 0.5 ␮g/mL ethidium bromide. The DNA fragment of 14.3
kb represents the amplified rat mtDNA, whereas the 2 fragments of
7.0 and 7.3 kb, which cannot be resolved and therefore migrate as a
single band on 1% agarose gel, represent the amplified mouse
mtDNA that contains an NcoI restriction site. The intensities of these
bands were assessed by image analysis (Molecular Dynamics)
followed by the quantitative densitometer with the software ImageQuant (version 3.3, Molecular Dynamics). The relative content of rat
mtDNA was derived by normalization with the mouse mtDNA in
each sample.
mtDNA in Focal Ischemia
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Alkaline Gel Electrophoresis and
Southern Blotting
Six micrograms of total DNA, including genomic and mitochondrial,
was digested with 10 U of restriction enzyme SacII (Promega) to
linearize mtDNA. After phenol/chloroform extraction and ethanol
precipitation, the concentrations of the DNA samples were determined by the PicoGreen method, as described above. Five micrograms of each sample was loaded onto a 1% agarose gel. Gel
electrophoresis was conducted at 0.5 V/cm for 24 hours in the buffer
containing 30 mmol/L NaOH and 2 mmol/L EDTA. The gels were
then neutralized, and DNA was transferred to Hybond N⫹ nylon
membrane by capillary blotting. The membrane was prehybridized
for 2 hours and hybridized overnight, both at 60°C, with the probes
labeled with digoxigenin by PCR. This probe specifically recognizes
the sequence of 16S ribosomal RNA (rRNA) in rat mtDNA. The
labeling of this 413-bp 16S rRNA fragment was accomplished with
the use of a PCR digoxigenin labeling/detection kit (Boehringer
Mannheim Corporation) with the primer pair containing the following sequences: 5⬘-TAGAATGAATGGCTAAACGAGG-3⬘ (forward) and 5⬘-TTAATAGCTTCTGCACCATTGG-3⬘(reverse).
Quantitative assessment of hybridization signal intensity was performed as previously described for LPCR.
Statistical Analyses
For comparisons between the left (control) and right (ischemic)
MCA cortices at each time point, Student’s unpaired 2-tailed t test
was used. For changes over time, 1-way ANOVA followed by Tukey
test was used. A value of P⬍0.05 was considered significant.
Results
Optimization of LPCR Conditions
A new strategy was applied in the quantitative LPCR to
assess mtDNA contents by including a robust internal control.
To optimize the experimental conditions for quantitative
LPCR, we first test the amplification cycles (Figure 1A) and
the amounts of template DNA (Figure 1B) to derive the
optimal cycles and optimal mtDNA output within the linear
ranges of the LPCR. A linear amplification pattern was
achieved from 24 to 30 PCR cycles (Figure 1A) or between
0.1 and 0.8 ng total DNA (Figure 1B). For consistency,
26-cycle and 0.4-ng total DNA were selected for the subsequent experiments.
LPCR for mtDNA Quantification
Results in Figure 1 demonstrate that mtDNA can be successfully amplified out of a small fraction of total DNA isolated
from the MCA cortex. However, the LPCR process could
amplify slight variations inherently present in each sample
and ultimately result in a substantial deviation in quantities of
amplified products, leading to potentially erroneous conclusions. To resolve this issue, we developed a new LPCR
approach to compare the relative rat mtDNA contents between different samples by including a known amount of
mouse template DNA in each sample as an internal control.
This approach is based on the high-sequence homology
between the mouse and rat mitochondrial genomes with
unique restriction enzyme sites not shared by the 2 species.
Thus, the amplified products of rat and mouse mtDNA can be
distinguished by a simple restriction enzyme digestion followed by gel electrophoresis. To ensure a linear correlation
between the amounts of rat total DNA input and the amplified
rat mtDNA in the presence of different amounts of the mouse
template, we conducted LPCR using a combination of vari-
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Figure 2. Validation of internal standards for LPCR. A, LPCR
was performed with a combination of different amounts of rat
and mouse templates. B, Representative LPCR assay of the relative mtDNA contents in the MCA cortices from rats subjected
to 30- or 90-minute ischemia with or without reperfusion (4 or
24 hours). The numbers indicate the ratio of relative mtDNA
contents between the ischemic right MCA cortex (R) and control
left cortex (L) after normalization with the internal standards
based on the mouse mtDNA template. Note that the ratio was
restored to near 1.0 at 4 and 24 hours after 30-, but not
90-minute, ischemia.
Figure 1. Optimization of experimental conditions for LPCR. A,
Linear range of LPCR cycles. Rat total DNA (0.4 ng) was amplified with a primer pair specific for mtDNA for 22 to 30 cycles.
Top, Ethidium bromide–stained agarose gel depicting the LPCR
product of 14.3 kb. M indicates DNA molecular weight marker.
Bottom, Correlation between the relative contents of amplified
LPCR products and cycle numbers as determined by densitometry scanning of the agarose gel. B, Linear range of DNA input.
Rat total DNA (0.1 to 0.8 ng) was amplified with mtDNA-specific
primers for 26 cycles. The agarose gel and linear relationship
between the amplified products and template inputs are shown
in top and bottom panels, respectively. Data shown are representative of 3 independent experiments with similar results.
able quantities of the rat and mouse templates to simultaneously amplify both mtDNA species. Despite the presence
of both species, the relative content of the LPCR product of
rat mtDNA is proportional only to the rat, but not the mouse,
DNA input (Figure 2A). Thus, a linear relationship between
the amounts of each template and the relative content of the
respective PCR product can still be established with PCR
mixtures containing DNA input from 2 different species
(Figure 2A). Further analysis by densitometry scanning
revealed that the amplification efficiencies are similar between the rat and mouse mtDNA within the linear ranges
defined in preliminary studies (data not shown). Together
these results suggest the feasibility to incorporate a known
amount of mouse template to normalize the relative content
of the rat LPCR product among different samples. A representative LPCR run to illustrate changes in mtDNA content in
ischemic rat brains is shown in Figure 2B. The animals were
subjected to either 30- or 90-minute focal ischemia followed
by reperfusion for 0, 4, or 24 hours. The relative mtDNA
content in the right MCA cortex was reduced by approximately half compared with the nonischemic left counterpart
immediately after ischemia for 30 or 90 minutes. The amplified mouse LPCR product serving as internal standard for
each sample remained unchanged. The relative mtDNA
content was restored to near nonischemic levels at 4 and 24
hours after ischemia for 30, but not 90, minutes.
Confirmation of LPCR Results by Southern
Blot Analyses
Southern blot analysis was implemented to further validate
this LPCR method. We conducted 30- and 90-minute ischemia with or without reperfusion for 24 hours. A portion of
total DNA prepared from these samples was subjected to
alkaline gel electrophoresis and probed with a labeled 16S
rRNA partial sequence in murine mitochondrial genome to
quantitatively assess the relative contents of intact mtDNA.
Another portion of the same DNA samples was subjected to
the LPCR assay. When the results derived from Southern
blotting (Figure 3A) and LPCR (Figure 3B) were compared,
a strong correlation between these 2 methods for mtDNA
quantitation could be established. Both approaches reveal that
the mtDNA content, which was reduced as a result of
30-minute ischemia, was fully restored after 24-hour reperfusion, whereas 90-minute ischemia caused a reduction in
mtDNA levels that was only partially restored at 24 hours.
These findings suggest that LPCR is capable of providing an
accurate, semiquantitative measure of the content of intact
mtDNA.
Reduction and Restoration of mtDNA Contents
After Focal Ischemia
To demonstrate that the LPCR assay of the mtDNA content is
reproducible, we conducted another study in which 6 animals
Chen et al
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Figure 3. Confirmation of LPCR results by Southern blot analysis. A, Rats were subjected to 30- or 90-minute ischemia with or
without 24 hours of reperfusion. A portion of the total DNA isolated from the ischemic right MCA cortex (R) or the nonischemic
left counterpart (L) was subjected to alkaline gel electrophoresis
followed by hybridization with a mitochondrial 16S rRNA partial
sequence labeled with digoxigenin, as described in detail in
Materials and Methods. B, The same DNA samples used for
Southern blotting were analyzed by LPCR. The numbers in the
bottom rows in both A and B denote ratios of the relative
mtDNA content in the ischemic right MCA cortex relative to the
nonischemic left counterpart. Note the close association
between relative mtDNA contents assessed by Southern blotting
vs LPCR from the same set of samples. Representative data
from 3 separate experiments with similar results are shown.
per group were subjected to either 30- or 90-minute ischemia
followed by reperfusion for 0, 0.5, 4, or 24 hours. In accord
with results shown in Figure 2B and Figure 3, 30-minute
ischemia caused a transient reduction of the relative content
of intact mtDNA at the end of ischemia, suggesting extensive
mtDNA damage that was partially restored as early as 30
minutes into reperfusion (Figure 4A). The relative mtDNA
content recovered to levels comparable to the contralateral
nonischemic counterpart after 4- or 24-hour reperfusion. In
contrast, while 90-minute ischemia caused similar reduction
in mtDNA contents, the restoration of mtDNA was not
complete up to 24 hours after reperfusion (Figure 4B).
Discussion
A number of mechanisms may underlie the mtDNA damages
after ischemic brain injury. The primary mechanism is likely
to be oxidative stress. A major function of mitochondria is
oxidative phosphorylation in the generation of energy supply
to sustain normal cellular functions. ROS are byproducts of
this important mitochondrial function. A higher steady state
level of oxidative damage was noted in mtDNA than in
nuclear DNA.3 Cerebral ischemia/reperfusion is accompanied
by an increase in brain ROS content.21,22 ROS may damage
mtDNA.4,23 Oxidative mtDNA damage may disrupt mitochondrial gene expression, resulting in mitochondrial dysfunction.24 Disturbances of mitochondria-specific gene expression occurred in the CA1 neurons at an early stage of
reperfusion, leading to progressive failure of energy production and ultimately neuronal death.25 Mitochondrial swelling
may also contribute to mitochondrial dysfunction. Cerebral
mtDNA in Focal Ischemia
2385
Figure 4. Reduction and restoration of relative mtDNA contents
after MCA ischemia/reperfusion. The relative mtDNA contents in
animals that had undergone 30-minute (A) and 90-minute (B)
ischemia without or with reperfusion for 0.5, 4, or 24 hours were
compared. The relative content of mtDNA in each ischemic right
MCA cortex (in percentage) was derived by using the nonischemic left MCA in the same animal as the denominator (100%).
Values are mean⫾SEM; n⫽6. *P⬍0.05 compared with the nonischemic side; #P⬍0.05 compared with other groups by 1-way
ANOVA followed by Tukey test.
hypoxia-ischemia has been shown to cause mitochondrial
swelling and calcium accumulation in the neonatal rat brain.26
Severe mitochondrial swelling may lead to the rupture of
mitochondrial membranes, with resultant release of mtDNA
that is susceptible to endonuclease digestion. Under such
circumstances it is likely that mitochondrial swelling may
contribute to the decreased mtDNA contents after ischemia/
reperfusion, which is an irreversible process. If this indeed
occurs in vivo, then such a reduction of mtDNA contents can
only be replenished with regeneration of mitochondria. In the
present study a decrease in mtDNA content was restored to
near control levels in the rats subjected to 30-minute, but not
90-minute, ischemia. It is therefore possible that short-term
ischemia did not result in irreversible mitochondrial dysfunction, allowing oxidative mtDNA damages to be repaired or
replenished. This may be one of the reasons why restoration
of mtDNA was observed only in the rat brains subjected to
30-minute, but not 90-minute, ischemia. Endonuclease activation may also cause mtDNA damage. Nuclear DNA fragmentation mediated by endonucleases is a common feature of
cells dying of apoptosis or necrosis and has been noted in the
ischemic brain after cerebral ischemia/reperfusion.16 Whether
mtDNA damage involves endonuclease action remains to be
determined.
After 30-minute ischemia, the mtDNA content was reduced initially but fully restored after 4- and 24-hour reperfusion. The recovery that began as early as 30 minutes after
reperfusion could be due to DNA repair, replication, or both.
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Measurement of mtDNA content based on the LPCR method
does not distinguish between mtDNA repair and mtDNA
replication. An increase in mtDNA content has been shown to
be the early molecular event of human cells in response to
mild oxidative stress such as exposure to hydrogen peroxide.27 It remains to be determined whether a change in
mitochondria number occurs in the rat brain cortices after
cerebral ischemia/reperfusion. Despite the changes in
mtDNA contents as determined by LPCR, it is important to
note that higher DNA content may not necessarily represent
higher DNA integrity and/or mitochondrial function. The
primer pair for LPCR was designed such that a PCR product
of 14.3 kb was amplified out of the total mtDNA, which is
16.3 kb. Since the amplified products cover approximately
88% of the whole mtDNA genome, we believe that it is a
reasonable index for the integrity of endogenous mtDNA
present in the rat brains after ischemia/reperfusion. It should
be noted, however, that higher DNA content may not represent higher DNA integrity and function.
At least 3 mechanisms may contribute to failure in restoring mtDNA content to nonischemic levels after 90-minute
ischemia. First, severe oxidative stress may render the damaged mtDNA irreparable. Severe ischemia/reperfusion can
induce acute tissue necrosis.13 Second, severe ischemic injury
may result in certain types of mtDNA damages that are
irreparable because of the lack of specific DNA repairing
mechanisms. For example, UV-induced pyrimidine dimers
are not removed from mtDNA since mitochondria do not
possess nucleotide excision repair machinery.28 Finally, irreversible release of mtDNA through ruptured mitochondrial
membrane secondary to mitochondrial swelling may occur
after severe ischemic brain injury. Electron microscopy may
be used to examine the ultrastructure of mitochondria at
subcellular level.
Previously LPCR has been shown to be a sensitive method
to assess the extent of DNA damage, repair, or replication, in
a semiquantitative fashion.29 In this study we report a new
approach using a simple and universally applicable internal
standard to reduce variations inherent to PCR. The highsequence homology shared by the target DNA (rat mtDNA)
and internal standard (mouse mtDNA) allows the concomitant amplification of both rat and mouse mtDNA using only
1 pair of primers with approximately similar efficiencies. The
end products derived from the target template are then
distinguished from the internal standard by NcoI restriction
enzyme digestion because the recognition sequence of NcoI is
only present in the mouse, but not rat, mtDNA. Similar
strategy can be applied to determine the mouse mtDNA levels
with the use of rat mtDNA as internal standards. A number of
control experiments were conducted to assess the reliability
and sensitivity of this LPCR approach. We optimized the
cycle numbers to be used such that maximal sensitivity may
be achieved without overamplification (Figure 1A). Pilot
experiments were conducted to establish a linear relationship
between the quantities of template DNA and those of LPCR
products (Figure 1B). When templates from 2 species were
amplified simultaneously, the amounts of LPCR products
yielded were only in proportion to the respective templates
(Figure 2A). Southern blot analysis coupled with alkaline gel
electrophoresis confirmed the LPCR results (Figure 3). This
LPCR method, however, carries certain limitations. For
examples, this method does not address the patterns of
oxidative mtDNA damage. Moreover, this LPCR technique
cannot provide spatial information with regard to the distribution of mtDNA lesions or changes in mtDNA content in a
specific area of brain cortex that may sustain irreversible
mtDNA damage and undergo apoptosis. Other in situ techniques will have to be developed to characterize the spatial
distribution of mtDNA lesions, including those at the subcellular level.
In conclusion, short-term (30-minute) ischemia caused
transient reduction in mtDNA content, which was nearly
restored to nonischemic levels within 4 hours of reperfusion;
longer duration of ischemia, causing a large infarct, was
associated with reduction in mtDNA content that was not
fully restored on reperfusion up to 24 hours. The mechanism(s) that restore mtDNA content after brief ischemia to
near nonischemic levels remains to be explored but may
involve mtDNA repair and/or repletion.
Acknowledgments
This work was supported by grants NS25545, NS28995, and
NS40525. We thank Dr Grace Ku for her critical review.
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Reduction and Restoration of Mitochondrial DNA Content After Focal Cerebral
Ischemia/Reperfusion
Hong Chen, Chaur-Jong Hu, Yong Y. He, Ding-I Yang, Jan Xu and Chung Y. Hsu
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Stroke. 2001;32:2382-2387
doi: 10.1161/hs1001.097099
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