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Evaluating therapeutic targets for reperfusion-related brain hemorrhage.

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Evaluating Therapeutic Targets for
Reperfusion-Related Brain Hemorrhage
Carolina M. Maier, PhD, Lily Hsieh, BS, Trisha Crandall, BS, Purnima Narasimhan, PhD, and
Pak H. Chan, PhD
Objective: Early reperfusion after an ischemic stroke can cause blood–brain barrier injury with subsequent cerebral edema and
devastating brain hemorrhage. These complications of early reperfusion, which result from excess production of reactive oxygen
species, significantly limit the benefits of stroke therapies. In this article, we use a novel animal model that facilitates identification of specific components of the reperfusion injury process, including vascular injury and secondary brain damage, and
allows assessment of therapeutic interventions.
Methods: Knock-out (KO) mice containing 50% manganese-superoxide dismutase activity (SOD2-KO) and transgenic mice
overexpressing SOD2 undergo transient focal ischemia and reperfusion followed by assessment of infarct, edema, hemorrhage
rates, metalloproteinase activation, and microvascular injury.
Results: SOD2-KO mice demonstrate delayed (⬎24h) blood–brain barrier breakdown associated with activation of matrix
metalloproteinases, inflammation, and high brain hemorrhage rates. These adverse consequences are absent in wild-type littermates and minocycline-treated SOD2-KO animals. Increased hemorrhage rates also are absent in SOD2 overexpressors, which
have reduced vascular endothelial cell death. Finally, we show that the tight junction membrane protein, occludin, is an early
and specific target in oxidative stress-induced microvascular injury.
Interpretation: This model is ideal for studying ischemia/reperfusion-induced vascular injury and secondary brain hemorrhage
and offers a unique opportunity to evaluate antioxidant-based neurovascular protective strategies as potential adjunct treatments
to currently approved stroke therapies such as thrombolysis and endovascular clot retrieval.
Ann Neurol 2006;59:929 –938
The World Health Organization (www.who.int) estimates that 15 million people suffer a stroke each year,
resulting in an annual mortality rate of 5 million and
an additional 5 million people suffering permanent disability.
Nearly 80% of strokes are due to occlusion of a cerebral artery by a thrombus. Early restoration of cerebral blood flow (reperfusion) can salvage hypoperfused
brain tissue, thus limiting neurological disability.
Reperfusion strategies have proved to be the most effective therapies for stroke treatment. In fact, the only
two stroke therapies approved by the US Food and
Drug Administration are a thrombolytic agent that can
dissolve occlusive thrombi (tissue-plasminogen activator) and a mechanical device that is capable of direct
retrieval of thrombi from within cerebral vessels (Merci
Concentric Retriever). One of the principal limitations
of these treatment strategies is that early reperfusion of
ischemic brain tissue can result in harmful consequences, including breakdown of the blood–brain barrier, which can lead to cerebral edema, brain hemorrhage, or both. Hemorrhages after reperfusion are
particularly devastating and associated with extremely
high morbidity and mortality. Fear of reperfusionrelated hemorrhage substantially limits the use of
stroke therapies; it is estimated that only 2 to 3% of
stroke patients in the United States receive acute reperfusion therapy.
The deleterious consequences of early restoration of
cerebral blood flow after stroke are attributed to reperfusion injury, a process that further damages brain
cells, the ischemic arterial wall, and the microvasculature.1–3 Yet, little is known about the pathophysiological mechanisms of reperfusion injury. This knowledge
gap stems, in part, from the lack of animal models to
facilitate identification of specific components of the
injury process and to allow evaluation of therapeutic
interventions.
Free radicals, specifically reactive oxygen species
(ROS) that are generated soon after vessel occlusion, as
well as in later stages of ischemic reperfusion (eg, by
inflammatory cells), are the fundamental mediators of
reperfusion injury. Here, we use an innovative animal
model to investigate the cellular and molecular events
From the Departments of Neurosurgery and Neurology and Neurological Sciences and Program in Neurosciences, Stanford University School of Medicine, Stanford, CA.
Published online May 3 2006 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.20850
Received Nov 8, 2005, and in revised form Feb 27, 2006. Accepted
for publication Mar 4, 2006.
Address correspondence to Dr Chan, Neurosurgical Laboratories,
Stanford University, 1201 Welch Road, MSLS P314, Stanford, CA
94305-5487. E-mail: phchan@stanford.edu
Published 2006 by Wiley-Liss, Inc., through Wiley Subscription Services
929
associated with the production of ROS and implicated
in ischemia/reperfusion-induced blood–brain barrier
disruption and intracerebral hemorrhage. Knock-out
(KO) mice with targeted disruption of the inducible
mitochondrial manganese-containing superoxide dismutase (SOD2) are subjected to a mild stroke followed
by early reperfusion and up to 3-day survival. These
heterozygous SOD2-KO mice (SOD2⫺/⫹) are more
susceptible to ischemic damage than their wild-type
(WT) counterparts and exhibit a significant increase in
matrix metalloproteinase-9 (MMP9) expression after
ischemia/reperfusion injury. The increase in MMP9
expression, which is particularly evident in blood vessels and inflammatory cells, precedes and is spatially
associated with a substantial increase in brain hemorrhage rates. The tight junction transmembrane protein
occludin is particularly susceptible to ROS- and
MMP9-induced damage in SOD2-KO mice and may
explain, in part, the increased microvascular injury observed in these animals. In contrast, animals overexpressing SOD2 (SOD2 transgenics [SOD2⫹/⫺]) and
SOD2-KO mice treated with minocycline, a broadspectrum antibiotic with antiinflammatory properties,
have significantly diminished hemorrhage rates, reduced infarct size, and improved survival.
Materials and Methods
The Stanford Institutional Administrative Panel on Laboratory Animal Care approved all animal protocols, and their
guidelines were followed for all procedures.
Focal Cerebral Ischemia and Drug Regimen
Heterozygous SOD2-KO mice4 (CD1/SV129 background,
backcrossed with CD1 for more than 10 generations), heterozygous transgenic mice overexpressing SOD25 (C57BL/6J
background, backcrossed with C57BL/6 for more than 10
generations), and their respective WT littermates were used.
Mice (32–35gm) anesthetized with 2% isoflurane (30% oxygen/70% nitrous oxide) and maintained at a surgical plane
of anesthesia (1–1.5% isoflurane) underwent middle cerebral
artery occlusion by the intraluminal suture method for 30
minutes (body temperature maintained at 37 ⫾ 0.5°C).
Reperfusion was allowed to occur in animals for 24 or 72
hours, and they had free access to food and water; then they
were killed with an isoflurane overdose at the desired end
point. For the drug study, half the mice (SOD2-KO animals
and WT littermates) received minocycline (day 1: at 1 and 4
hours after insult, 45mg/kg intraperitoneally; days 2 and 3:
single dose of 22.5mg/kg), whereas the other half received
equal amounts of saline.
Histopathology, Perl’s Iron Staining,
Immunohistochemistry, and Terminal
Deoxynucleotidyl Transferase Deoxyuridine
Triphosphate Nick End Labeling Staining
Annals of Neurology
Vol 59
No 6
Evans Blue Extravasation
Six hours before death, 2.5ml/kg of 4% Evans blue (Sigma)
in 0.9% saline was injected into every animal. Animals were
anesthetized and transcardially perfused with 200ml heparinized saline followed by 3.7% paraformaldehyde. Brains were
removed, fixed overnight in 3.7% paraformaldehyde, and
sectioned with a vibratome at 20␮m. Sections were incubated (10 minutes) with 2␮g/ml Hoechst 33258 (Molecular
Probes, Carlsbad, CA) for nuclear counterstaining and
mounted, and blood–brain barrier integrity was assessed by
fluoromicroscopic detection of Evans blue extravasation.
Western Blot Analysis
After transcardiac perfusion with heparinized saline (10U/ml)
followed by 3.7% paraformaldehyde, the brains were removed,
930
postfixed (3 days), paraffin-embedded, sectioned (6␮m-thick
coronal sections), and stained with hematoxylin and eosin. Infarct was evaluated in the hemisphere, cortex, and striatum;
histological criteria included areas of pan-necrosis with
shrunken dark neurons and glial pallor. Infarct areas (seven
coronal levels6) were determined using an image-analysis system (MCID; Imaging Research, Linton, United Kingdom)
and corrected for edema.7 Diaminobenzidine-enhanced Perl’s
iron staining was conducted in deparaffinized tissue by incubation in 1% KFeCN/1% HCl (15 minutes) followed by
methyl green counterstain. For immunofluorescence, frozen
brain sections treated for endogenous peroxidases and blocked
for avidin-biotin were used. Sections were incubated with primary antibody (MMP9, anti-rabbit and anti-human, 1:100
[Chemicon, Temecula, CA]; NeuN [Chemicon]; glial fibrillary acidic protein [GFAP; Sigma, St. Louis, MO]; factor VIII
[Research Lab, South Bend, IN]; myeloperoxidase [Dako,
Denmark]; CD11b, 1:200; 4°C, 1 hour [Accurate, Westbury,
NY]) followed by secondary antibody (1:200; 1-hour paraffin
sections; 10-minute frozen sections; 25°C). In the paraffinembedded sections, MMP9 incubation lasted 72 hours; antibodies were detected using a Vector-ABC kit and colorized
with Vector-VIP (Vector Laboratories, Burlingame, CA). Vascular structures and microglia were identified with biotinylated
isolectin-B4 (IB4; 3-h incubation; Vector); reaction products
were detected with Vector-ABC kit and colorized with diaminobenzidine. Both colorizing agents were also used for identification of different cell types or for colocalizing protein expression through double labeling. Fluorescein avidin-DCS,
Texas-Red Avidin-D, or AMCA Avidin-D was used for frozen
sections; tissue was mounted with 4⬘,6 diamidino-2-phenylindole–containing medium. Negative control samples were run
in parallel using adjacent sections incubated without a primary
antibody.
Terminal deoxynucleotidyl transferase deoxyuridine
triphosphate nick end labeling staining was performed using
the ApopTag peroxidase system (Intergen, Burlington, MA).
In brief, sections were deparaffinized, treated with proteinase
K (20␮g/ml for 15 minutes) and for endogenous peroxidases, and subjected to incubation with terminal deoxynucleotidyl transferase for 1 hour (37°C). Sections were
washed, then incubated with antidigoxigenin peroxidase conjugate for 30 minutes, followed by peroxidase substrate, additional washes, and counterstaining with methyl green.
June 2006
Each hemisphere was homogenized in suspension buffer (1M
KOH, 1M KCl, 1M MgCl2, 0.5 EDTA, 0.5M EGTA, su-
crose, protease cocktail) and centrifuged (14,000g, 10 minutes,
4°C), and supernatants were transferred into equal volumes of
sodium dodecyl sulfate buffer with ␤-mercaptoethanol and
stored (⫺20°C). Aliquots were subjected to sodium dodecyl
sulfate polyacrylamide gel electrophoresis (10 –20% Trisglycine gel; Invitrogen, Carlsbad, CA). Polyvinylidene difluoride membranes were incubated in methanol, then in transfer
buffer (5 minutes each). Transfer (25V, 1.5 hours) was followed by incubation in 5% milk in phosphate-buffered salineTween (60 minutes). Membranes were incubated overnight
(4°C) in primary antibody (listed earlier plus hemoglobin [Research Plus, Manasquan, NJ], aquaporin-4 [AQP4; Chemicon], occludin, claudin-5, zonula occludens-1 [Zymed, San
Francisco, CA]; 1:100 dilution, 1% milk in phosphatebuffered saline-Tween) and then in peroxidase-conjugated secondary antibody (1:200; 1 hour, 25°C). Signals were detected
with a chemiluminescence kit enzyme chemiluminescence
(ECL)-plus Western Blotting Detection System (GE Healthcare, Piscataway, NJ). To confirm consistent protein loading
for each lane, we stained membranes for ␤-actin. Films were
scanned with a GS-700 imaging densitometer (Bio-Rad, Hercules, CA); quantitative analyses were performed using MultiAnalyst software (Bio-Rad).
Microvessel Isolation
Brains (n ⫽ 10 per isolation) were obtained from mice subjected to ischemia/reperfusion as described earlier. Meninges,
large surface vessels, and corpus callosum were removed and
discarded. The tissue was then minced and digested with collagenase/dispase (Roche, Basel, Switzerland) for 1 hour and
centrifuged at 1,000g for 5 minutes. The pellet was resuspended in 20% BSA and centrifuged at 3,000g for 10 minutes and digested in collagenase/dispase/DNAse containing
M199 medium for 1 hour. This was then washed, resuspended in Earle’s Balanced Salt Solution (without phenol
red) and centrifuged in a 50% Percoll gradient at 3,000g for
10 minutes. The band containing microvessels was collected,
resuspended in 6M urea lysis buffer with a protease inhibitor
cocktail overnight, then centrifuged, and protein concentrations were determined8,9 (with the above modifications).
Mouse Cerebral Endothelial Cell Culture
Cerebral cortices of 3-week-old WT and SOD2⫺/⫹ mice
were isolated and then treated as described earlier with some
modifications. The band containing microvessels was collected and plated onto collagen-coated dishes in endothelial
cell medium with supplements (BD Biosciences, San Jose,
CA) and grown to confluence before use.
Treatment of Cultures with Oxygen-Glucose
Deprivation
The endothelial cells from both WT and SOD2⫺/⫹ mice
were subjected to oxygen-glucose deprivation by replacing
the medium in the cell culture plates with buffered salt solution without glucose and placing the cell culture plates in
an anaerobic chamber (Plas Labs, Lansing, MI) at 37°C for 8
hours. The oxygen-glucose deprivation period was terminated by replacing the buffered salt solution with endothelial
cell medium and placing the cell culture plates in the
5%CO2/95% air incubator for different reoxygenation periods. Two wells were examined per condition.
Analysis of Brain Vasculature
The integrity of the circle of Willis of SOD2⫺/⫹ and
SOD2⫹/⫺ mice and their respective littermates was examined by carbon black injection. The animals were anesthetized with isoflurane and perfused through the left ventricle
with heparinized saline and subsequently with a mixture of
carbon black and 10% gelatin. The brain was removed and
fixed in 3.7% paraformaldehyde for 24 hours. The cerebral
vasculature was photographed with a dissecting microscope.
Statistical Analyses
Statistical analyses were done with one-way analysis of variance followed by two-tailed Student’s t test (two-group comparisons) and with the nonparametric Wilcoxon signed rank
test for noncontinuous data. All analyses were made with
Stat-View 4.0 software (SAS Institute, Cary, NC). Data in
the text and figures are expressed as mean ⫾ standard error
of the mean; p less than 0.05 was considered significant.
Results
Superoxide Dismutase-2 Knock-out Mice Are Highly
Susceptible to Ischemic Brain Injury, Edema, and
Hemorrhage after Ischemia/Reperfusion
We subjected SOD2-KO mice and their WT counterparts to 1 hour of middle cerebral artery occlusion followed by reperfusion and established that the mortality
rate for the SOD2-KO animals increased from 10% at
24 hours to nearly 90% at the 72-hour end point. In
contrast, the 72-hour mortality rate in the WT animals
was only 10%. Examination of the brain specimens
showed a clear correlation between the mortality rate
and the occurrence of large intracerebral hemorrhages
in SOD2-KO animals. This was in sharp contrast with
heterozygous SOD1-KO mice, which had a mortality
rate (63% at 96 hours) that was associated with severe
cerebral edema but not hemorrhage. We then reduced
the ischemic period to 30 minutes and demonstrated
that the SOD2-KO mortality rate was reduced to 30%
at 72 hours. Histopathology showed that these animals
had a statistically significant increase in infarct size at
all levels examined (Fig 1A) at both 24 and 72 hours
compared with the WT mice, as well as an increase in
the rate of hemorrhagic transformations (SOD2-KO:
8/9; WT: 3/8) at 72 hours, which was not observed at
24 hours (see Fig 1B). These hemorrhagic transformations were generally widespread throughout the ischemic tissue and often quite large in the SOD2-KO
mice (see Figs 1B, C), and many were observed in the
borderzone between infarct and surviving tissue. In
contrast, the few (and typically small) hemorrhagic
transformations observed in the WT littermates were
found predominantly in the ventral portion of the ischemic hemisphere, near the entry point of the middle
Maier et al: Reperfusion-Related Hemorrhage
931
Fig 1. Reperfusion-induced brain injury and hemorrhage and the effects of minocycline treatment. After 30 minutes of middle cerebral artery occlusion, superoxide dismutase-2 knock-out (SOD2-KO; black bars) animals had a statistically significant increase in
infarct size at all levels examined (A; *p ⬍ 0.05, analysis of variance) at both 24 and 72 hours of reperfusion compared with
wild-type (WT; white bars) littermates. The use of 2,3,5-triphenyltetrazolium chloride to delineate infarcts showed that SOD2-KO
animals had increased intracerebral hemorrhage rates at 72 hours of reperfusion, which were absent at 24 hours, as well as in most
of the WT littermates (B). These results were confirmed by Western blot analysis (B, bottom), which showed increased brain hemoglobin values, indicating intracerebral hemorrhage, in the SOD2-KO mice compared with WT littermates at 72 hours of reperfusion (optical density measurements for SOD2-KO: 174 ⫾ 34; WT: 29 ⫾ 18; p ⫽ 0.009, Student’s t test). Additional confirmation came from Perl’s iron staining (brown stain with methyl green counterstain), which showed that hemorrhagic transformations,
although generally widespread throughout the ischemic tissue (C), were often observed in the borderzone between infarct and surviving tissue (dashed line delineates the infarct). In addition, several SOD2-KO mice (5/9) had large hemorrhagic transformations
(D) associated with microvascular leakage (left inset) and activated macrophages (right inset). SOD2-KO mice also had a significant increase in cerebral edema compared with the WT littermates at the 72-hour end point (E; *p ⫽ 0.018, Student’s t test);
which was associated with a significant increase in aquaporin-4 (AQP4) expression (F; *p ⫽ 0.006, Student’s t test). Treatment
with minocycline reduced infarct size at the 72-hour end point in the SOD2-KO mice and WT littermates at all levels examined
compared with saline treatment (G; *p ⬍ 0.05, analysis of variance). Perl’s iron staining also demonstrated a reduction in hemorrhagic transformations in the minocycline-treated SOD2-KO mice (H). Ctx ⫽ cortex; Hem ⫽ hemisphere; St ⫽ striatum. Scale
bar ⫽ 20␮m.
cerebral artery, suggesting that these lesions resulted
from mild mechanical damage to the occluded vessel.
Brain edema, which plays a critical role in the pathophysiology and morbidity of stroke, was increased by
55% in SOD2-KO mice compared with the WT littermates at the 72-hour end point ( p ⫽ 0.018; see Fig
1E). The increase in cerebral edema in these animals
was associated with a significant increase in AQP4 expression ( p ⫽ 0.006; see Fig 1F), a water-selective
transporting protein that is abundantly expressed at the
blood–brain barrier (in perivascular and endothelial
cells) and at the interface of brain tissue and cerebrospinal fluid.10 –12 We previously have shown that
AQP4 deletion in mice reduces brain edema after an
ischemic stroke.13 Whether the increase in AQP4 expression after ROS-induced reperfusion injury is involved in exacerbating edema formation or occurs as
the brain’s attempt to facilitate clearance of excess water remains to be determined.
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June 2006
We previously have shown that oxidative stress is
involved in mediating blood–brain barrier disruption
during the first 3 to 7 hours of reperfusion after an
ischemic event through MMP activation.14 In addition, by comparing SOD2-KO with WT control animals, we demonstrated that after 1 hour of middle
cerebral artery occlusion, SOD2 reduces apoptotic
cell death.15 Moreover, SOD2 attenuates cytochrome
c release from the mitochondria to the cytosol, a critical step in the intrinsic mitochondrial-dependent signaling pathway of the cell death program.16 These
findings, in conjunction with the remarkably increased 72-hour hemorrhage rate observed in the
SOD2-KO animals, led us to hypothesize that ischemia/reperfusion caused increased ROS production in
mitochondria, leading to MMP activation, blood–
brain barrier breakdown, vasogenic edema, and delayed ischemic neuronal damage.
Minocycline Reduces Infarct Size, Hemorrhage Rate,
Mortality, and Matrix Metalloproteinase-9 Expression
To test our hypothesis and validate our model, we
treated SOD2-KO mice and their WT littermates with
minocycline, a commonly used tetracycline derivative
that crosses the blood–brain barrier and reduces cytochrome c release from mitochondria17 and also has
been shown to attenuate stroke severity.18 Minocyclinetreated mice (SOD2-KO and WT littermates) had a significant reduction in infarct size at 72 hours of reperfusion compared with saline-treated animals (see Fig 1G).
In addition, minocycline treatment reduced the hemorrhage rate in SOD2-KO mice (3/11 vs 7/9 treated with
saline; see Fig 1H), as well as the mortality rate (2/13 vs
4/13 treated with saline). Because reactive microglia pro-
duce MMP919 and minocycline has been shown to reduce microglial activation,20 we examined the effects of
minocycline on MMP9 expression in our model. We
found that minocycline significantly reduced MMP9 expression in SOD2-KO mice at 24 and 72 hours of
reperfusion (Fig 2), with the relative reduction being
particularly significant at the latter time point (75% relative reduction at 72 hours; see Fig 2D).
Superoxide Dismutase-2 Knock-out Mice Have
Increased Matrix Metalloproteinase-9 Expression in
the Vascular Endothelium
The ischemia/reperfusion-induced increase in blood–
brain barrier disruption has been linked to inflammatory events that involve ROS production and
Fig 2. Effects of minocycline on ischemia/reperfusion-induced matrix metalloproteinase-9 (MMP9) expression. Western blot analysis
with a rabbit anti–mouse MMP9 antibody (105kDa band) in whole-brain homogenates showed that minocycline (Min) treatment
significantly reduced MMP9 expression in superoxide dismutase-2 knock-out (SOD2-KO) mice at 24 (*p ⫽ 0.03) and 72 hours
(†p ⫽ 0.05) of reperfusion compared with saline (Sal) treatment (A), although this effect was not significant in the wild-type
(WT) littermates at 72 hours of reperfusion (B). We also tested a rabbit anti–human MMP9 antibody (C). Pro-MMP9 was evident as 92kDa characteristic bands in the saline- and minocycline-treated animals, whereas activated MMP9 was detected as
88kDa bands. Similar results were obtained from independent studies (n ⫽ 3) for A and B. (D) The overall relative reduction in
MMP9 expression with minocycline treatment (from three separate experiments).
Maier et al: Reperfusion-Related Hemorrhage
933
leukocyte-endothelial cell adhesion and migration. This
is supported by studies that show that agents targeting
either the generation of ROS by endothelial cells
and/or the adhesion of leukocytes to vascular endothelium are generally effective in blunting the ischemia/
reperfusion-induced increase in microvascular permeability.21 We recently determined that leukocytes,
specifically neutrophils that comprise more than 50%
of circulating leukocytes, are not the primary source of
MMP9 protein in the brain even at the peak of leukocyte infiltration (24 –72 hours after stroke onset)6 and,
thus, are unlikely to be the key contributor to the
blood–brain barrier breakdown observed in SOD2-KO
mice. In contrast, MMP9 immunoreactivity in vascular
structures (found throughout the infarcted hemisphere
but most prominently in the borderzone between infarct and surviving tissue) was significantly stronger in
SOD2-KO mice compared with WT littermates. To
further study the relationship among ROS production,
MMP9 expression, and blood–brain barrier disruption,
we injected Evans blue into the jugular vein of the
SOD2-KO and WT mice after ischemia/reperfusion.
Extravasation of Evans blue from vessels, which indicates vascular permeability, was detected throughout
the ischemic hemisphere (Fig 3, red fluorescence).
MMP9 immunofluorescence (see Fig 3, green fluorescence) was easily discerned in vessels showing Evans
blue extravasation. MMP9 expression was particularly
prominent in small vessels in the infarct border (see Fig
3B), but could also be observed in vascular structures
within severely damaged areas in the center of the infarct, the ischemic core (see Fig 3C). MMP9immunoreactive cells were also observed in the corpus
callosum (see Figs 3D, E), as well as in larger vessels
such as the middle cerebral artery (see Figs 3F, G).
Reactive Oxygen Species Mediate Increases in
Endothelial Matrix Metalloproteinase-9 and Damage
to Microvascular Endothelial Tight Junctions
The brain endothelial cells that form the blood–brain
barrier have tight junctions that are critical for maintaining brain homeostasis and restricting permeability.
Some of the major components of tight junction complexes are zonula occludens-1, occludin, and claudin-5,
all of which are targets of MMP9 degradation. The
volume of the brain capillary endothelium forming the
blood–brain barrier is approximately 10⫺3 parts of the
whole brain,22 thus making detection of small changes
Fig 3. Spatial relationship between Evans blue extravasation and matrix metalloproteinase-9 (MMP9) expression. Representative
photomicrographs show colocalization of Evans blue (red fluorescence) and MMP9 (green fluorescence) in a superoxide dismutase-2
knock-out (SOD2-KO) animal subjected to 30 minutes of focal cerebral ischemia and 72 hours of reperfusion. Extravasation of
Evans blue from vessels was detected throughout the ischemic territory (A). MMP9 immunofluorescence was evident in small vessels
showing Evans blue extravasation in the infarct border (B) where small hemorrhages could be detected (arrow and inset) and
within severely damaged areas in the center of the infarct, the ischemic core (C). MMP9 immunoreactivity was also observed in the
corpus callosum (D), although vessels in this region were not always immunoreactive (E; inset shows the negative control), as well as
in larger vessels such as the middle cerebral artery (F, G). Scale bar ⫽ 20␮m.
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in tight junction components difficult in whole-brain
homogenates. To address this issue, we adapted a microvessel isolation technique that allows us to isolate
free radical–mediated blood–brain barrier–specific
events after ischemia/reperfusion injury. To generate
sufficiently large protein concentrations for the Western blots, we pooled a total of 10 brains for each isolation procedure (1 lane ⫽ 10 brains). The end product (microvessel pellet) was tested for factor VIII (an
endothelial cell marker), NeuN (a neuronal marker),
and glial fibrillary acidic protein, a glial marker, to ensure successful isolation (Figs 4A, B).
As expected, we found progressive degradation of
zonula occludens-1, occludin, and claudin-5 after ischemia/reperfusion injury. The SOD2-KO animals were
particularly sensitive to occludin degradation and
showed an associated increase in MMP9 expression
(see Fig 4C) compared with WT littermates (see Fig
4C). Results from five separate experiments showed
that MMP9 expression in SOD2-KO animals was, on
average, 21% greater at 24 hours and 14% greater at
72 hours than in WT littermates.
To confirm our MMP9 results from the microvessel
isolations, we also tested the effects of oxygen-glucose
deprivation on endothelial cell cultures from the
SOD2-KO and WT mice. After 8 hours of oxygenglucose deprivation, which resulted in 50% cell death,
and 0 to 8 hours of reoxygenation, endothelial cells
from the SOD2-KO animals showed a marked increase
in MMP9 expression compared with endothelial cell
cultures from the WT littermates (see Fig 4E). By 24
hours of reoxygenation, there was a reduction in
MMP9 levels in the SOD2-KO endothelial cell cultures that was inversely proportional to cell death in
those cultures. WT endothelial cell cultures, in contrast, showed a substantial increase in MMP9 levels
and only a small increase in cell death at the same time
point.
Superoxide Dismutase-2 Protects against Blood–Brain
Barrier Damage after Ischemia/Reperfusion Injury
As proof of principle, we subjected transgenic mice
overexpressing SOD2 (which have a 2.5-fold increase
in SOD2 activity) and their WT littermates to the
same ischemia/reperfusion paradigm. Histopathology
and Perl’s iron staining showed that the rate of hemorrhagic transformations at 72 hours was quite low in
both groups (SOD2 transgenic: 3/9; WT: 3/9) and was
restricted mostly to the entry point of the middle cerebral artery and, thus, likely due to mechanical damage by the occluding filament. Because genetic mutations can affect vessel anatomy and, hence, stroke
outcome, we analyzed the brain vasculature of the mice
by carbon black injections. All animals showed an intact circle of Willis (Fig 5), indicating that there were
no differences in gross vascular anatomy among the
SOD2-KO mice, transgenic animals overexpressing
SOD2, and their respective WT counterparts. However, when we examined the microvasculature within
the infarcted area, we observed qualitatively more cells
stained positive for terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling in
microvessels of the SOD2-KO mice compared with
their WT littermates, whereas such cells in the transgenic animals overexpressing SOD2 were rare.
Discussion
SOD2-KO mice and the newly developed transgenic
mouse overexpressing SOD2 provide a unique opportunity to elucidate, in a molecular fashion, the oxidative mechanisms that mediate brain injury after stroke
and mitochondria-related blood–brain barrier dysfunctions. Furthermore, SOD2-KO animals can facilitate
the development and evaluation of treatment strategies
that diminish the complications of cerebral reperfusion.
This is particularly important given the approaches being investigated to improve stroke treatment. One
strategy for increasing the number of patients eligible
for thrombolysis consists of delaying the progression of
stroke by delivering high-flow oxygen to patients before vessel recanalization.23 Oxygen therapy can enhance the formation of ROS and, thus, increase blood–
brain barrier damage, but recent work suggests that this
therapy may extend the reperfusion window when
treatment is administered during the ischemic episode.24 The effects of combining oxygen therapy with
thrombolytic agents or clot retrieval devices have not
yet been determined.
In this study, we observed that SOD2-KO mice had
a high rate of reperfusion-related brain hemorrhage,
larger infarct volumes, and cerebral edema. We hypothesized that these hemorrhages and the amplification of ischemic injury were caused by increased production of ROS in mitochondria, leading to activation
of MMPs, blood–brain barrier breakdown, vasogenic
edema, and delayed ischemic neuronal damage. To test
our hypothesis, we treated SOD2-KO mice with minocycline, an agent with a primary mechanism of action that is a direct inhibition of cytochrome c release
from mitochondria, followed by inhibition of downstream cell death–related mechanisms.17 Minocycline
treatment led to reduced hemorrhage rates, infarct volumes, and mortality. Although we cannot exclude the
possibility that the reduction in microvascular injury
was due, in part, to a reduction in infarct size, our
findings demonstrate that pharmacological agents have
the potential to attenuate reperfusion-related brain injury and that our model can be used to evaluate putative therapeutic agents.
To clarify the mechanisms underlying the increased
brain injury observed in the SOD2-KO mice, we developed a unique technique for microvessel isolation af-
Maier et al: Reperfusion-Related Hemorrhage
935
Fig 4. Microvessel isolations and endothelial cell cultures after ischemia/reperfusion injury. Western blot analysis of two representative microvessel isolation experiments (A and B are the graphic representations of the Western blots shown in D). After 30 minutes of middle cerebral artery occlusion and 24 or 72 hours of reperfusion, microvessels were isolated from superoxide dismutase-2 knock-out (SOD2-KO)
mice and wild-type (WT) littermates (n ⫽ 10 per group for each isolation). The microvessel pellets were tested for factor VIII, NeuN, and
glial fibrillary acidic protein (GFAP) to ensure a clean isolation. Although some variability was observed, our results showed little contamination from either neuronal or glial cells. The samples then were tested for zonula occludens-1 (ZO-1), occludin, and claudin-5 expression,
and our results showed progressive degradation of all three proteins during evolution of the infarct. The SOD2-KO animals were particularly sensitive to occludin degradation (A, B). (C) The optical density (OD) measurements of MMP9 expression from optimal microvessel
isolation. The SOD2-KO mice showed an increase in MMP9 expression compared with WT littermates at 24 and 72 hours of reperfusion. (E) We obtained similar results when we subjected cerebral capillary endothelial cell cultures from the SOD2-KO and WT mice to 8
hours of oxygen-glucose deprivation, which resulted in 50% cell death, followed by reoxygenation. Endothelial cells from the SOD2-KO
animals showed a marked increase in MMP9 expression at the end of the ischemic period and up to 8 hours of reoxygenation. By 24
hours, MMP9 levels declined in the SOD2-KO endothelial cells, which paralleled a concomitant increase in cell death in those cultures.
MMP9 expression in the WT endothelial cell cultures did not increase until 24 hours of reoxygenation, at which point most cells were still
viable. WB ⫽ whole brain.
936 Annals of Neurology Vol 59 No 6 June 2006
Fig 5. Vascular integrity as assessed by carbon black injections and terminal deoxynucleotidyl transferase deoxyuridine triphosphate
nick end labeling (TUNEL) staining. Analysis of the brain vascular anatomy showed an intact circle of Willis in the superoxide
dismutase-2 knock-out mice (SOD2⫺/⫹) and SOD2 overexpressors (SOD2⫹/⫺) and their respective WT littermates (A–D, top).
Representative photomicrographs from animals subjected to 30 minutes of middle cerebral artery occlusion and 24 hours of reperfusion (middle and bottom rows). The SOD2-KO mice showed increased TUNEL staining (brown; methyl green counterstain)
throughout the infarcted area compared with WT littermates. Arrowheads indicate TUNEL-positive endothelial cells in small vessels. TUNEL immunoreactivity was rarely seen in vascular structures from the SOD2 transgenic mice. Scale bar ⫽ 20␮m.
ter in vivo ischemia/reperfusion. This technique allowed us to verify the existence of blood–brain barrier
injury and demonstrate that this injury was likely mediated by an increase in MMP9 expression. Furthermore, this technique allowed us to establish that, in
endothelial cells, one of the key consequences of mitochondrial oxidative stress and the ensuing overexpression of MMP9 is damage to the tight junction transmembrane protein occludin. This finding supports
previous work demonstrating that occludin can be selectively cleaved by metalloproteinases,25 and that
phosphorylation of occludin appears to be a key regulator of tight junction permeability.26
Finally, although reducing the capacity of mitochondria to scavenge superoxide radicals in endothelial cells
increases susceptibility to damage and death, we were
able to demonstrate that overexpression of SOD2 has a
protective effect on the microvasculature. Whether
this increase in mitochondrial antioxidant capacity
can enhance cell-survival mechanisms is a crucial
question that is currently under investigation. The
availability of transgenic mice overexpressing SOD2
allows us to perform direct comparisons of the microvascular responses between these mice and SOD2-KO
mice after ischemia and, thus, to examine the specific
molecular and cellular mechanisms that underlie
ischemia/reperfusion-related brain injury. The techniques described here will facilitate these discoveries
and ultimately translate into new therapeutic interventions for stroke patients.
This work was supported by American Heart Association Postdoctoral Fellowship (0120142Y, C.M.M.), the NIH (National Institute
of Neurological Disorders and Stroke, NS 14534, NS 25372, NS
36147, NS 38653, P.H.C.), and an American Heart Association
Bugher Foundation Award (P.H.C.).
We thank R. Sobel for expert advice, T.-T. Huang and A. Richardson for helping us set up the SOD2 transgenic colony, T.P. Davis
and collaborators for their expert advice in microvessel isolation procedures, G.W. Albers for his expert advice and editorial assistance,
P. Bracci for statistical analysis, C. Christensen for editorial assistance, and E. Hoyte for preparation of the figures.
Maier et al: Reperfusion-Related Hemorrhage
937
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