Stroke. 1998;29:1020-1030
(Stroke. 1998;29:1020-1030.)
© 1998 American Heart Association, Inc.
Matrix Metalloproteinase Expression Increases After Cerebral Focal Ischemia in Rats
Inhibition of Matrix Metalloproteinase-9 Reduces Infarct Size
Anne M. Romanic, PhD;
Raymond F. White, BS;
Anthony J. Arleth, BS;
Eliot H. Ohlstein, PhD;
Frank C. Barone, PhD
From the Department of Cardiovascular Pharmacology, SmithKline Beecham
Pharmaceuticals, King of Prussia, Pa.
Correspondence to Dr Anne M. Romanic, Department of Cardiovascular Pharmacology, UW2510, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd, King of Prussia, PA 19406. E-mail anne_romanic-1{at}sbphrd.com
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Abstract
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Background and Purpose—Matrix
metalloproteinases (MMPs) are a family of proteolytic enzymes that
degrade the extracellular matrix and are implicated in numerous
pathological conditions including atherosclerosis,
inflammation, and tumor growth and metastasis. In the brain, the
endothelial cell wall, strengthened by tight junctions,
defines the blood-brain barrier (BBB). The extracellular matrix
molecules constitute the basement membrane underlying the vasculature
and play a critical role in maintaining the integrity of the BBB. After
focal stroke, there is a breakdown of the BBB with an associated
increase in vascular permeability, inflammatory cell influx, and
neuronal cell death. The present study was designed to investigate
the effects of MMP expression after stroke.
Methods—Focal stroke was produced by permanent middle cerebral
artery occlusion (MCAO) in the rat, and MMP protein expression was
measured by Western blot and zymogram analysis over a time
course ranging from 6 hours to 30 days (n=32). Immunohistochemistry at
1 and 5 days (n=8 and 6, respectively) was also utilized to
characterize the expression of several MMPs and related proteins after
stroke, including their cellular source. To test the hypothesis that
early increased MMP-9 expression is involved in ischemic brain
injury, a neutralizing monoclonal antibody directed against MMP-9 was
administered intravenously (n=7 per group) 1 hour before
MCAO, and infarct size was measured 24 hours later.
Results—MMP expression increased progressively over time after
stroke. After 12 hours, significant (P<0.05) MMP-9
activity was observed that reached maximum levels by 24 hours
(P<0.001), then persisted for 5 days at this level and
returned to basal (zero) levels by 15 days. On the basis of
morphological criteria, MMP-9 appeared to stain with
endothelial cells and neutrophils identified both
within and at the periphery of the infarct within 24 hours of focal
ischemia. After 5 days, MMP-9 appeared to stain with
macrophages present within the infarcted brain. MMP-2
activity was significantly (P<0.001) increased by 24
hours and was maximum after 5 days following MCAO. MMP-2 appeared to
stain with macrophages present within the infarcted region.
Unlike MMP-9 and MMP-2, tissue inhibitor of
metalloproteinase-1 was identified at comparable levels in both control
and ischemic tissue after MCAO. MMP-1 and MMP-3 could not be
detected in the brain after focal stroke. When an MMP-9–neutralizing
monoclonal antibody was administered systemically, animals exhibited
significantly reduced infarct size (ie, a 30% reduction compared with
non–immune antibody controls; P<0.05).
Conclusions—These results demonstrate that early increased MMP-9
expression in endothelial cells and infiltrating
neutrophils is a significant response to cerebral focal
ischemia and that selective inhibition of MMP-9 activity can
significantly reduce brain injury after stroke.
Key Words: antibody inhibition • blood-brain barrier • endothelium, vascular • leukocytes • metalloproteinases • neutrophils • stroke, ischemic
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Introduction
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The ECM is a
multifunctional complex of proteins and proteoglycans assembled in a
highly organized manner that contributes to the structural integrity of
cells and tissue within an organ system. The basement membrane, which
provides structural support to the vasculature, is composed of ECM
molecules such as type IV collagen, laminin, and
fibronectin.1 Various factors are involved in
maintaining the integrity of the ECM and the tissues it supports. However, in
certain pathological circumstances the ECM is modulated such that the
structure of the tissue becomes damaged or destroyed. MMPs are a group
of zinc-dependent enzymes that degrade the molecules of the
ECM.2 3 4 Two members of the MMP family, MMP-2
(72-kD gelatinase/gelatinase A) and MMP-9 (92-kD gelatinase/gelatinase
B), degrade the ECM components of the basement membrane. Their
substrates include types IV and V collagen, fibronectin, elastin, and
denatured interstitial collagens. Matrix degradation
attributed to these proteinases has been shown to play an important
role in the progression of diseases such as
atherosclerosis,5 6
inflammation,7 8 and tumor growth and
metastasis.9 10 11
In the brain, the endothelial cell wall,
strengthened by tight junctions, defines the
BBB.12 The basement membrane underlying the
vasculature plays a critical role in maintaining the integrity of the
BBB by providing structural support to the endothelial
cell wall.1 12 The BBB serves to protect the
central nervous system from invasive agents, such as inflammatory cells
and bacteria, as well as from chemical agents. After focal stroke there
is a breakdown of the BBB with an associated increase in vascular
permeability. Damage to the BBB often results in hemorrhage and
edema, resulting in neuronal cell death.13 14
Brain injury after focal stroke is primarily a result of the decrease
in blood flow and of energy depletion due to occlusion of a cerebral
blood vessel. The neuronal tissue becomes infarcted as a result of
these events, with contributions from excitotoxicity, enzyme
activation, edema, and inflammation.15 16
It has been demonstrated that MMP-2 injections directly into rat brain
disrupt the BBB and are associated with the increased expression of
MMP-9.17 More recently, an increase in MMP
activity after stroke has been demonstrated.18
Related studies have shown that laminin, fibronectin, and type IV
collagen (all basement membrane components) are lost after cerebral
ischemia and reperfusion, contributing to the loss of
microvascular integrity.19 There is also a
significant inflammatory response that follows stroke. It has been
shown that leukocytes, including neutrophils and macrophages,
infiltrate into the ischemic brain
tissue20 21 and contribute to ischemic
brain injury.22 Furthermore, it has been
demonstrated that neutrophils utilize MMPs for their
migration.23 These studies suggest a role for
MMPs and basement membrane degradation in the breakdown of the BBB and
in the leukocyte infiltration that occurs with stroke.
The cellular source(s) of MMP expression and its contribution to focal
ischemic injury have not previously been studied. The
present series of experiments were performed to define the time
course, isozyme subtype, and cellular sources of increased MMP
expression that occurs after cerebral focal ischemia in the
rat. In addition, a selective MMP-9 inhibitor was
administered systemically to rats receiving cerebral ischemia
to determine whether selective, early blockade of increased MMP-9
activity could reduce brain injury after focal stroke.
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Materials and Methods
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Focal Brain Ischemia
Cerebral focal ischemia or sham surgery was performed in
male spontaneously hypertensive rats (Taconic Farms, Germantown, NY) at
16 to 18 weeks of age weighing 250 to 330 g by permanent MCAO, as
described in detail previously20 21 24 and in
compliance with the guidelines for the care and use of laboratory
animals approved by The Animal Care and Use Committee at SmithKline
Beecham Pharmaceuticals. Briefly, the animals were anesthetized
with pentobarbital (60 mg/kg IP), and the MCA was occluded and cut
dorsal to the lateral olfactory tract at the level of the
inferior cerebral vein with the use of electrocoagulation
(Force 2 Electrosurgical Generator, Valley Laboratory Inc). In
sham-operated rats the dura was opened over the MCA, but the artery was
not occluded. Body temperature was maintained at 37°C until recovery
from anesthesia. Rats were later killed with an overdose of
pentobarbital, and the forebrains were removed for cortical dissection
at various times after MCAO. In some cases the ischemic
frontoparietal cortex was dissected from the ipsilateral hemisphere and
the contralateral cortex was dissected as the nonischemic
control from the same rat.21 Cortical samples
were frozen immediately and stored at -80°C until used for protein
analysis (see below). Coronal sections were also made through
the forebrain, and the tissue was prepared for
immunohistochemistry20 (see below). The time
points selected for observation after stroke (6, 12, and 24 hours and
5, 15, and 30 days) in the present study were based on earlier
histological characterization of this model in terms of
ischemic injury, cellular infiltrate, and resolution of tissue
damage.20 Ipsilateral cortical samples from
sham-operated animals killed at 12 and 24 hours and the contralateral
cortex of all operated focal ischemic rats were compared.
Several animals (n=4 to 8) were included at each time point under each
condition for comparison in each of the techniques that are described
below.
Preparation of Tissue Extracts
To analyze protein expression patterns in control and
ischemic brain tissue, protein extracts of the tissues were
prepared. The forebrains were removed from each animal at various times
after surgery, and cortical samples were dissected as described above.
Immediately after dissection, the tissues were stored at -80°C until
all the samples from the time course were collected. To prepare the
tissues for extraction, they were first weighed and then minced into
1-mm3 pieces. The minced tissues were incubated
in an extraction buffer consisting of 0.5% Triton X-100 (Sigma) in PBS
containing 0.5 U/mL aprotinin (Sigma) and 0.01% sodium azide while
gently rotating at 4°C for 18 hours. The concentration of the initial
extraction mixture for each tissue sample was normalized to 500 mg/mL.
After the extraction was complete, the samples were centrifuged
at 14 000 rpm for 10 minutes at 4°C, and the supernatants were
collected, aliquoted into 10-µL volumes, and stored at -20°C. To
check the quality and uniformity of each extraction throughout the
study (ie, to demonstrate that the extractions were consistent
between time points and animals evaluated), samples of each extract
prepared were analyzed by SDS-PAGE (10%
polyacrylamide) in which the gel was stained with 0.25%
Coomassie brilliant blue R-250 (Sigma).
Western Blot Analysis
To investigate the protein expression of various MMPs in control
and ischemic tissue extracts, equal volumes (10 µL) of tissue
extracts normalized for protein concentration were prepared for Western
blot analysis. Briefly, extracts were resolved by
electrophoresis through a 10% polyacrylamide gel under
reducing conditions25 and then transferred to a
nitrocellulose membrane. Unoccupied binding sites were blocked
overnight at 4°C with 5% nonfat powdered milk in a 0.1 mol/L
Tris-HCl buffer, pH 8.0, containing 1.5 mol/L NaCl and 0.5% Triton
X-100 (TBST buffer). A primary antibody, diluted in TBST, was then
added to the membrane and allowed to incubate for 1 hour at 25°C. The
membrane was washed three times, 15 minutes each, with TBST and then
incubated for 30 minutes with a secondary antibody conjugated to
horseradish peroxidase (Sigma). The membrane was washed as above, and
the blot was developed with the use of the enhanced chemiluminescence
method (Amersham) according to the manufacturer's instructions. The
primary antibodies used included the following: mouse monoclonal
antibodies directed against MMP-1 (clone 41-IE5, Oncogene Science),
MMP-2 (clone 42–5D11, Oncogene Science), and MMP-3 (clone 55–2A4,
Oncogene Science); rabbit polyclonal antibodies directed against MMP-3
(Biogenesis); and MMP-9, a gift from Dr Hideaki Nagase, University of
Kansas Medical Center.
SDS-PAGE Zymography
MMP enzyme expression was assayed by zymography as
described by Herron et al.26 Briefly, equal
volumes (10 µL) of tissue extracts normalized for protein
concentration were subjected to electrophoresis, without boiling or
reduction, through a 10% polyacrylamide gel copolymerized with
gelatin (0.5 mg/mL) or casein (0.5 mg/mL) at 4°C. After
electrophoresis was complete, the gel was incubated for 1 hour at
25°C in a 2.5% Triton X-100 solution, washed two times, 20 minutes
each, with water, and then incubated overnight at 37°C in a 0.05
mol/L Tris-HCl buffer, pH 8.0, containing 5 mmol/L
CaCl2. As a control, duplicate samples were
loaded onto another gel that was then incubated in a 0.05 mol/L
Tris-HCl buffer, pH 8.0, containing 10 mmol/L EDTA to inhibit MMP
activity. The gels were fixed with 40% methanol and 7% acetic acid,
stained with 0.25% Coomassie blue R-250, and then destained with 10%
methanol and 7% acetic acid. Enzyme activity attributed to MMP-1,
MMP-2, and MMP-9 can be visualized (on the basis of molecular weight)
in the gelatin-containing zymograms as clear bands against a blue
background. Similarly, casein-containing zymograms can be used to
determine MMP-3 activity. To quantify the relative levels of MMP
expression as detected by SDS-PAGE zymography, the gels were digitized,
and the area of lysis for each band detected was quantified by
computer-assisted planimetry of the lytic zone area in square
millimeters (Amersham RAS 3000 Image Analysis System, Loats
Associates, Inc).
In Situ Zymography
Although SDS-PAGE zymograms are useful in identifying the
presence of latent ("pro") and active forms of various MMPs, they
cannot indicate the actual net proteolytic activity because of the
presence of TIMPs. TIMPs are generally coexpressed with MMPs in tissue
samples and can inhibit MMP activity. However, the presence of SDS in
SDS-PAGE zymograms displaces TIMPs from the MMPs and also
activates latent enzymes. Therefore, to analyze net
endogenously active MMP expression within the brain tissue
after MCAO, in situ zymography was conducted. With this method,
endogenous MMP activity could be identified and correlated
to a particular region of the tissue. Brain tissue from animals that
had undergone focal cerebral ischemic or sham surgery was
removed after 24 hours and immediately placed on ice. Coronal sections
(1 mm) were made through the forebrain and rinsed briefly in PBS.
The sections were then directly overlaid on top of a gel consisting of
10% polyacrylamide copolymerized with gelatin (0.5 mg/mL) in
50 mmol/L Tris, 5 mmol/L CaCl2, pH 7.4.
The tissue sections were incubated on top of the gel for 8 hours at
37°C, after which the sections were removed and the gel was incubated
further for a total of 18 hours. The gels were fixed with 40% methanol
and 7% acetic acid, stained with 0.25% Coomassie blue R-250, and then
destained with 10% methanol and 7% acetic acid. Gelatinase activity
was visualized as a zone of clearing against a blue background.
Immunohistochemistry
Forebrains were removed from animals that had undergone
permanent focal cerebral ischemia after 24 hours (n=8) or 5
days (n=6) and immediately placed on ice. Sham-operated rats killed
after 24 hours (n=5) or 5 days (n=5) were used as controls. Coronal
sections (2 mm) were made through the forebrain, after which the
sections were fixed with 10% (wt/vol) phosphate-buffered formalin
(Baxter Scientific Products) for 18 to 20 hours. After standard
histological processing and embedding in paraffin,
6-µm-thick sections were prepared for immunoperoxidase staining with
the Vectastain Elite ABC kit (Vector Laboratories) according to the
manufacturer's instructions. Briefly, endogenous
peroxidase was quenched with 0.3%
H2O2 in methanol for 30
minutes. Nonspecific immunoglobulin binding sites were blocked with
normal goat serum for 1 hour, and then the sections were incubated with
a primary monoclonal antibody for 1 hour at room temperature. As a
negative control, serial sections were incubated with mouse IgG (Vector
Laboratories) instead of the primary antibody. The sections were then
incubated for 30 minutes with a biotinylated goat anti-mouse IgG
secondary antibody (1:200, Vector Laboratories) followed by 30 minutes
of incubation with the Vectastain Elite ABC reagent solution.
Immunoglobulin complexes were visualized on incubation with DAB (Vector
Laboratories) at 0.5 mg/mL in 50 mmol/L Tris-HCl (pH 7.4) and 3%
H2O2. DAB staining was
enhanced by treating the sections for 10 seconds with DAB Enhancing
Solution (Vector Laboratories). Sections were washed, counterstained
with Gill's hematoxylin, cleared, mounted with Aquamount
(Polysciences), and then examined by light microscopy with an Olympus
IX70 microscope. The primary monoclonal antibodies used for these
studies were as follows: anti–MMP-1 (clone 41-IE5, Oncogene Science),
anti–MMP-2 (clone 42–5D11, Oncogene Science), anti–MMP-3 (clone
55–2A4, Oncogene Science), anti–MMP-9 (clone 56–2A4, Oncogene
Science), anti–TIMP-1 (clone 7–6C1, Oncogene Science),
anti–neurofilament-200 (N52, Sigma), and an antibody that recognizes
monocytes and macrophages (clone ED1, BioSource).
In Vivo Studies
Spontaneously hypertensive rats (Taconic Farms), weighing 300 to
330 g, were treated with a murine neutralizing monoclonal antibody
previously demonstrated to inhibit MMP-9
activation.27 The IgG monoclonal antibody (clone
6–6B, Oncogene Science) was dissolved in sterile PBS, and an estimated
inhibitory dose was administered intravenously
(3 mg/kg) 1 hour before MCAO. As an appropriate control for comparison,
nonimmune mouse IgG (Vector) was administered (3 mg/kg) in the same
manner to another group of animals.
MCAO was performed as described above; 24 hours later the animals were
killed with an overdose of pentobarbital, and the forebrains were
sectioned into seven coronal slices and immersed in a 1% solution of
2,3,5-triphenyltetrazolium chloride in a
1.0 mol/L phosphate buffer, pH 7.0, at 37°C for 20
minutes.28 Stained tissue slices were digitized
and analyzed as described previously20 24
with the use of computerized assisted planimetry (Optimus Image
Analysis, Inc and customized software). Infarct measurements
were adjusted for brain swelling due to edema, which is known to
contribute to and overestimate the degree of
infarction.29 30 Infarct area in square
millimeters was expressed as the percent infarcted tissue relative to
the contralateral normal hemisphere area in square millimeters. In
addition, the total volume of forebrain infarction was calculated from
the infarct areas on the individual forebrain slices.
Statistical Analysis
Data were expressed as mean±SEM. For statistical
analysis of gel lytic zone data, an ANOVA was followed up with
the Fisher's least significant difference test. For percent
hemispheric infarct and infarct volume measurements, the t
test for unpaired data was used. Statistical significance was accepted
at P<0.05.
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Results
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MMP-9 and MMP-2 Are Expressed in Ischemic Brain Tissue
Extracts
The time course of MMP protein expression after occlusion of the
MCA was assessed in tissue extracts prepared from control and
ischemic regions of rat brain. The proteins extracted from the
tissues were evaluated by SDS-PAGE before further analysis, and
the quality of the extracts was demonstrated to be satisfactory (data
not shown). MMP-1, MMP-2, MMP-3, and MMP-9 protein expression were
evaluated by Western blot. The results demonstrated that MMP-9 was
detected in the ischemic tissue within 24 hours after occlusion
and was still observed at the 5-day time point (Figure 1A
). By day 15, however, MMP-9 protein
expression was no longer observed. MMP-9 expression was absent in the
contralateral control cortex samples at each time point
analyzed (Figure 1A). MMP-2 expression was also detected in the
ischemic tissue but predominantly at the 5-day time point
(Figure 1B). MMP-9 and MMP-2 were not detected in the sham-operated
animals (data not shown). Western blot analysis was conducted
for MMP-1 and MMP-3 protein expression, but these proteins were not
detected in any of the samples assayed (data not shown).
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Figure 1. Western blot analysis of stroke-induced
MMP-9 and MMP-2 in ischemic rat brain tissue extracts. Tissue
extracts were analyzed for stroke-induced MMP-9 and MMP-2
expression over a time course of 6 hours to 30 days by Western blot
analysis. Blot A, MMP-9 expression detected at the 24-hour and
5-day time points. Blot B, MMP-2 expression detected at the 5-day time
point. Lanes marked with A indicate samples prepared from the
ischemic regions of the rat brain. Lanes marked with B indicate
samples prepared from the contralateral control regions of the rat
brain. Data are representative examples; similar
analyses were conducted in other animals with identical results
(n=4 animals per time point studied).
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MMP expression was also evaluated by SDS-PAGE zymography with the use
of gels containing either gelatin or casein. The results of these
experiments showed that MMP-9 activity was detected 6 hours after
stroke, was significantly increased 12 hours after stroke, was markedly
(maximally) expressed at 24 hours, and remained maximally expressed
after 5 days (Figure 2A). The expression
of MMP-9 returned to basal (zero) level at the 15-day time point. On
the basis of molecular weight, the results suggested that MMP-9 was
predominantly in the active form (molecular weight=87 kD) with only a
modest amount present as the latent "pro" form (molecular
weight=92 kD) (Figure 2A). Furthermore, it was demonstrated that MMP-2
occurred in both the latent and active forms and was identified in the
ischemic cortical samples (Figure 2A). However, while MMP-9
activity was detected after 6 hours and significantly expressed 12
hours after stroke, MMP-2 expression was detected after 24 hours but
was most pronounced (ie, maximal) 5 days after injury and was still
detectable at nonsignificant low levels for up to 30 days after MCAO
(Figure 2A). Figure 2B illustrates these quantified results for MMP-9
and MMP-2 after sham surgery and at various times after MCAO compared
with the contralateral control and sham samples in which MMP expression
was not detected. MMP-1 expression was not detected in the
gelatin-containing zymograms. Likewise, MMP-3 expression was not
detected in the casein-containing zymograms (data not shown). For
Western blot analysis and SDS-PAGE zymography, four
different sets of tissue samples collected over the entire time course
were analyzed (ie, four animals per time point). The results
shown in Figures 1
and 2A are representative of all the
sets evaluated.
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Figure 2. SDS-PAGE zymographic analysis of
stroke-induced MMP activity in ischemic rat brain tissue
extracts. A, Tissue extracts were analyzed for stroke-induced
MMP activities over a time course of 6 hours to 30 days with the use of
SDS-PAGE zymograms containing either gelatin or casein. Active MMP-9
(molecular weight [MW]=87 kD) and MMP-2 (MW=67 kD) as well as the
latent "pro" forms (MW=92 kD and 72 kD, respectively) were detected
in the gelatin-containing zymogram shown here. Lanes marked with A
indicate samples from the ischemic cortical region of the rat
brain. Lanes marked with B indicate samples from the contralateral
control cortical region of the rat brain. MW markers are as indicated
on the left. Data are representative examples; similar
analyses were conducted in other animals with identical results
(n=4 animals per time point studied). B, Quantified data for MMP-9
(solid bars) and MMP-2 (open bars) expression in ischemic
cortex tissue extracts as detected by SDS-PAGE zymography based on the
technique describe in "Materials and Methods." Statistical
significance was determined by ANOVA followed by the Fisher's least
significant difference test. Measurements for all animals studied were
made, including all sham samples and every time point/condition
indicated. Comparisons are with ipsilateral sham cortex samples or all
other sample (time) groups. No enzyme activity was detected in either
hemisphere of sham surgery rats or in the contralateral hemisphere of
MCAO rats. Some of the samples collected after MCAO exhibited
negligible amounts of activity and appear as "baseline" values in
the figure (ie, are zero or near zero and had no/little standard
errors). n=4 animals per time point.
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Net MMP Activity Is Detected by In Situ Zymography in the
Ischemic Hemisphere of the Rat Brain 24 Hours After
MCAO
Since MMPs can be present in either a latent or active form
and can be coexpressed with TIMPs, we were interested in identifying
the net MMP activity within the rat brain after stroke. To
analyze brain tissue samples for net MMP activity, in situ
zymography was conducted. Coronal brain slices were prepared from
animals 24 hours after undergoing focal cerebral ischemia or
sham surgery and then analyzed for
gelatinolytic activity by zymography. The 24-hour
time point was chosen for these studies to determine the net
proteolytic activity resulting from increased MMP expression by the
time brain infarction was maximum after stroke. Figure 3A depicts representative
zymograms indicating MMP activity detected only in the ischemic
hemisphere (closely associated with the cortical infarction
characterized in this model previously24 ) in
animals that had undergone MCAO (n=6) and not in the contralateral
control cortical region. In addition, Figure 3B illustrates that
ipsilateral cortical MMP activity was not detected in brain slices
prepared from animals that had undergone sham surgery (n=4).
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Figure 3. Induced MMP activity is detected by in situ
zymography in the ischemic rat brain 24 hours after MCAO. Net
MMP activity was evaluated in coronal brain sections prepared from
animals that had undergone focal cerebral ischemic or sham
surgery. MMP activity was visualized as a zone of clearing associated
with the ischemic region of the rat brain corresponding to the
infarcted region produced in the model. A, Brain sections from animals
that had undergone MCAO and were killed 24 hours later. B, Brain
sections from animals that had undergone sham surgery and were killed
24 hours later. Sections taken from two different animals that were
representative for each condition are shown. Arrows
indicate gelatinolytic activity detected in the
cortex of the ischemic hemisphere of the rat brain. Results are
representative of the total number of animals evaluated
(n=6 for MCAO and 4 for sham surgery).
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MMP-9 Is Observed With Endothelial Cells and
Neutrophils in Ischemic Brain Tissue 24 Hours After
MCAO
MMP-9 expression was evaluated by immunohistochemical
analysis in brain tissue sections prepared from animals that
had undergone surgery to induce focal cerebral ischemia or sham
surgery. The tissues were collected 24 hours after surgery (n=8), the
time point at which MMP-9 expression was elevated while MMP-2
expression was only moderately expressed as determined by SDS-PAGE
zymography (Figure 2). At this time point, animals that had
MCAO-induced focal ischemia exhibited significant cortical
tissue damage (Figure 4) and contained
infiltrated neutrophils (Figures 5 and 6), consistent with earlier
observations.20 22 Diffuse MMP-9 expression was
observed within and at the periphery of the ischemic cortical
lesion (Figure 4B). In contrast, there was no detectable MMP-9
expression in either control contralateral regions of the brain (Figure 4A) or in tissue from rats that had undergone sham surgery (n=5) (data
not shown).
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Figure 4. Immunohistochemical analysis of MMP-9
expression induced in ischemic rat brain 24 hours after MCAO.
Coronal brain sections were analyzed for MMP-9 protein by
immunohistochemistry. MMP-9 expression was not observed in
contralateral control brain but was identified within and at the
periphery of the ischemic cortical region, including corpus
callosum and some striatum, 24 hours after occlusion of the right MCA.
A, Contralateral control (magnification x10); B, ischemic
region (magnification x10). Representative section
illustrates MMP-9 immunoreactivity identified in 24-hour MCAO rats (n=8
studied). No labeling was identified in ipsilateral or contralateral
cortices of sham-operated rats (n=5; data not shown). C indicates
cortex; S, striatum (caudate nucleus and putamen); and CC, corpus
callosum. Note that A and B are matched photographs from the same
rat.
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Figure 5. Induced MMP-9 expression is observed with
endothelial cells in ischemic rat brain 24
hours after MCAO. Endothelial cells in microvessels
both within and at the periphery of the ischemic region appear
to express MMP-9 24 hours after infarct as determined by
immunohistochemistry. Higher-power photomicrographs of area similar but
more dorsal to that depicted in Figure 4 identify cellular elements
immunoreactive with the anti–MMP-9 antibody. A, Ischemic
region (magnification x25); B, area identified by box in panel A
(magnification x100). Thin arrows indicate morphologically identified
neutrophils that appear to express MMP-9; thick arrows indicate
morphologically identified endothelial cells that
appear to express MMP-9. Note that MMP-9 is detected in the
endothelial wall of the microvessel in cross section
and not present within the lumen. Representative
section illustrates labeling identified in rats (n=8 studied) 24 hours
after MCAO. No labeling was identified in control contralateral brain
after MCAO or in ipsilateral or contralateral cortices of sham-operated
rats (n=5; data not shown). C indicates cortex; S, striatum (caudate
nucleus and putamen); and CC, corpus callosum.
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At sites located both within and at the periphery of the
ischemic lesion, immunoreactive MMP-9 appeared to stain with
endothelial cells (identified by morphometric criteria)
of microvessels (Figure 5). In addition, MMP-9 expression appeared to
present with a significant number of neutrophils (identified by
morphometric criteria) that had migrated into the damaged cortical
region of the ischemic tissue (Figures 5 and 6). No similar
labeling of neutrophils or endothelial cells was
observed in either cortex after sham surgery (data not shown). Serial
sections that were incubated with mouse IgG instead of MMP-9 primary
antibody were negative (data not shown).
MMP-9 and MMP-2 Expression Is Observed With Macrophages in
Ischemic Brain Tissue 5 Days After Cerebral Infarct
The expressions of MMP-9 and MMP-2 were examined by
immunohistochemistry in rat brain isolated 5 days after MCAO.
Consistent with the observations made by SDS-PAGE zymography,
MMP-9 and MMP-2 expressions were detected 5 days after MCAO (n=6) in
the ischemic cortical region of the rat brain (Figure 7B and 7D, respectively) and were absent
in the control contralateral region (Figure 7A and 7C, respectively).
MMP-9 and MMP-2 were not observed 5 days after sham surgery (n=5, data
not shown). In addition, with the use of an antibody that recognizes
monocytes and macrophages (ED1), it was demonstrated that MMP-9
and MMP-2 appeared to stain with macrophages (identified by
morphometric criteria; identical morphology for MMPs and ED1) resident
within the ischemic lesion (Figure 7B, 7D, and 7F). Although
the ED1 antibody also stains activated microglial cells, the
morphology of the ED1-positive cells observed within the lesion was
indicative of macrophages and not microglial cells. Serial
sections that were incubated with mouse IgG instead of MMP-9 or MMP-2
primary antibodies were negative (data not shown).
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Figure 7. Induced MMP-9 and MMP-2 are observed with
macrophages in ischemic rat brain 5 days after MCAO.
Representative photomicrographs depict MMP-9 and MMP-2
expression identified in the ischemic regions of the rat brain
5 days after MCAO (n=6 rats studied) but absent in the contralateral
control regions. Note the significant deterioration of the
ischemic cortex 5 days after MCAO. A, MMP-9, contralateral
control (magnification x25); B, MMP-9, ischemic region
(magnification x25); C, MMP-2, contralateral control (magnification
x25); D, MMP-2, ischemic region (magnification x25); E, ED1,
contralateral control (magnification x25); F, ED1, ischemic
region (magnification x25). Insets, Enlarged regions indicated by tip
of arrows (magnification x100). MMP-9 and MMP-2 appear to be expressed
by morphologically identified macrophages resident within the
infarct 5 days after MCAO, as also determined by staining with a marker
for macrophages, ED1. No labeling was identified in
ipsilateral or contralateral cortices of sham-operated rats (n=5; data
not shown). C indicates cortex; CC, corpus callosum. Note that A, C,
and E are photographs matched to B, D, and F, respectively, from the
same rats.
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|
TIMP-1 Expression Is Similar in Both Control and Ischemic
Brain Tissue
Immunohistochemical analysis was conducted to investigate
the presence of TIMP-1 in control and ischemic rat brain in
tissues prepared 24 hours (n=8) and 5 days (n=6) after MCAO. The
results of these studies demonstrated that TIMP-1 expression was
localized in both the control and ischemic sides of the brain
(Figure 8A, 8B, and 8C) after MCAO and
that the levels of expression were comparable to that observed in brain
tissue from animals that had undergone sham surgery (n=5 per time
point) (data not shown). In addition, the levels of TIMP-1 expression
remained relatively unchanged at both the 24-hour and 5-day time points
(Figure 8B and 8C). Furthermore, TIMP-1 was present not only in the
cortical region of the brain but also in the nerve tracks (corpus
callosum and striatum [caudate nucleus and putamen]) located within
the white matter and, as determined by staining of serial
sections, appeared to have a staining pattern similar to that of a
marker for neurofilaments, NF-200, indicating axonal labeling within
these nerve tracks (Figure 8D, 8E, and 8F).
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Figure 8. Immunohistochemical identification of TIMP-1
expression in control and ischemic rat brain.
Representative photomicrograph indicates that TIMP-1 is
constitutively expressed in the ischemic and the contralateral
control brain regions of the rat brain 24 hours (n=8 studied) and 5
days (n=6 studied) after MCAO and stains with a pattern similar to that
of a marker for neurofilaments, NF-200, suggesting neuronal/axonal
localization. A, TIMP-1, contralateral control, 24 hours (magnification
x25); B, TIMP-1, ischemic region of same rat as in panel A, 24
hours (magnification x25); C, TIMP-1, ischemic region, 5 days
(magnification x25); D, NF-200, ischemic region, 24 hours
(magnification x25); E, TIMP-1, ischemic region, 24 hours
(magnification x100); F, NF-200, ischemic region, 24 hours
(magnification x100). TIMP-1 expression was constitutively expressed
in both cortices of sham-operated rats (n=5; data not shown). S
indicates striatum (caudate nucleus and putamen); CC, corpus
callosum.
|
|
Administration of an MMP-9 Inhibitor Reduces
Infarct Size
Intravenous treatment with the neutralizing monoclonal
antibody inhibitor of MMP-9 (3 mg/kg) 1 hour before MCAO
significantly reduced percent hemispheric infarct size by 28.3%
(Figure 9A; P<0.05) and
infarct volume by 29.9% (Figure 9B; P<0.05) compared with
the mouse IgG control. Previous studies have demonstrated that in using
an IgG antibody therapeutic agent against tumor necrosis
factor-
,31 administration of an immune or
nonimmune IgG antibody into this animal model does not exhibit any
effects on blood pressure, body temperature, blood gases, pH, or blood
glucose levels.
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Figure 9. Reduction in infarct size 24 hours after MCAO in
rats due to inhibition of MMP-9 by treatment with an MMP-9–blocking
antibody. Treatment with an MMP-9–blocking monoclonal antibody (3
mg/kg IV) administered 1 hour before MCAO significantly decreased
infarct size. A, Percent hemispheric infarct; B, infarct volume.
Anti–MMP-9 monoclonal antibody, n=7; mouse IgG treatment, n=7.
*P<0.05, t test.
|
|
|
Discussion
|
|---|
The present studies indicate that after focal
ischemia there are marked differences in the distribution and
temporal expression of MMP-9 and MMP-2 at the ischemic site.
MMP-9 expression was detected much earlier after focal ischemia
and appeared to stain with many endothelial cells and
infiltrating neutrophils. While not all neutrophils appeared to stain
for MMP-9 after 24 hours, it is possible that most individual
neutrophils might express MMP-9 associated with their migration at some
point after stroke. The later (ie, 5 days after stroke) expression of
MMP-9 and MMP-2 appeared to stain predominantly with
macrophages. Expression of MMP-9 by endothelial
cells within and at the periphery of the ischemic lesion
appears to be a key mechanism by which the cerebral vascular walls
become compromised, leading to edema and leukocyte infiltration. It has
been demonstrated that endothelial cells secrete MMPs,
including MMP-9 in the basal direction, thereby facilitating the
degradation of the basement membrane.32
Proteolysis of the ECM components of the basement membrane underlying
the vasculature has been associated with an increase in vascular
permeability and the loss of vascular
integrity.19 Additionally, degradation of the
vascular basal lamina allows the blood vessels to become permissive to
neutrophil extravasation into the brain tissue. In addition, as
neutrophils migrate from the circulation into the tissue, it has been
reported that they employ MMP-9 for their
invasion.23 The later expression of MMP-2 with
MMP-9 by macrophages may aid in their migration into the
ischemic lesion as well and might also contribute to the
clearing of cellular debris later during the later
wound-healing/resolution phase after focal
stroke.20
MMP expression can be modulated by various mechanisms. Regulation of
MMP expression can occur not only at the level of transcription but
also at the level of enzymatic activation.2 3 4
MMPs are secreted in a zymogen form containing a propeptide that
renders the enzyme inactive. Notably, active MMP-9 was identified in
ischemic tissue in the present study. The results of the in
situ zymography clearly demonstrated this. Activation of MMP-9 has been
demonstrated to occur on proteolytic cleavage by MMP-3, or
stromelysin,33 34 although other unknown
mechanisms of activation are still possible. We speculate that since we
were unable to detect MMP-3 in the ischemic tissue using two
different antibodies directed against MMP-3, MMP-9 is activated
by another unknown mechanism. However, another possibility is that we
were unable to identify a transient expression of MMP-3 in the early
phase of the time course.
MMP expression can also be regulated by enzyme inhibitors.
The endogenous inhibitors of MMP-9 and MMP-2
are the TIMPs, with TIMP-1 having preferred selectivity for inhibiting
MMP-9, and TIMP-2 having preferred selectivity for inhibiting
MMP-2.2 3 4 In diseases in which excessive matrix
degradation occurs, the balance between MMPs and TIMPs is often offset,
resulting in an overall net increase in MMP
activity.35 The results of the
immunohistochemical analysis demonstrated that TIMP-1 was
expressed at comparable levels in both the control and ischemic
cortical regions of the brain. Interestingly, TIMP-1 expression was
also present within the white matter of the corpus callosum
bordering the infarcted cortex. The expression pattern was similar to
that of a marker for neurofilaments in the nerve tracks present
within this white matter. These same regions were completely devoid of
MMP expression. It could be hypothesized that TIMP-1 provides an
endogenous means of protecting the nerve tracks from
structural damage due to MMP proteolysis.
The role of MMP-9 in stroke was examined further by administering a
neutralizing anti–MMP-9 monoclonal antibody to animals before MCAO in
the present study. The results of this experiment demonstrated that
the selective, early inhibition of MMP-9 significantly reduced the
degree of brain infarction, clearly demonstrating the importance of
this enzyme in contributing to focal ischemic brain injury.
Breakdown of the BBB after stroke can result in hemorrhage and
edema and ultimately, because of lack of blood flow, neuronal cell
death.13 14 15 16 The protection provided by MMP-9
inhibition can be due to blocking the early actions of MMP-9 secreted
by the endothelial cells. However, MMP-9 expression by
neutrophils can also contribute to their migration and exacerbation of
ischemic brain injury.22 36 It is
unlikely that the neuroprotective effects are mediated by IgG antibody
effects on vascular or blood parameters since previous
thorough evaluation of these parameters demonstrated no
effects of IgG antibodies.31
In summary, increased MMP-9 and MMP-2 expressions were induced in
ischemic regions of the rat brain and were not detected in
nonischemic control samples. Given the potential complexity of
central nervous system–associated MMPs,37 it may
be premature to focus on MMP-9 and MMP-2 in ischemic stroke.
However, the present data substantiate the importance of these two
enzymes in early and late phases of ischemia, respectively. The
data also corroborate previous studies demonstrating an increase of
these two MMPs after stroke18 and extend beyond
those studies by identifying the cellular sources of MMP-9 and MMP-2
and demonstrating neuroprotection produced by interfering with MMP-9
activation. In our assays, MMP-9 expression was markedly expressed
within 12 to 24 hours after MCAO, and its expression appeared to stain
with endothelial cells and neutrophils identified both
within and at the periphery of the ischemic lesion. After 5
days, MMP-9 expression was also detected with macrophages
within the infarct. Compared with MMP-9, MMP-2 expression was detected
later after MCAO, perhaps associated with the resolution (ie, the
remodeling and healing processes that occur after stroke). Like MMP-9,
MMP-2 was detected with macrophages within the infarct at the
5-day time point. Finally, the expression of TIMP-1, an
inhibitor of various MMPs, was evaluated and was shown to
be similar in both the control and ischemic brain tissue. This
stroke-induced MMP expression can be expected to shift the
protease-protease inhibitor balance toward proteolytic
activity and ECM degradation. In particular, the expression of MMP-9
within the first 24 hours after stroke suggested that it is involved in
the initial tissue destruction and contributes to the rapidly
developing brain injury that occurs after stroke. Systemic pretreatment
with an anti–MMP-9 neutralizing antibody produced a significant
decrease in brain injury observed 24 hours after stroke. Although
additional studies need to be performed to evaluate dose-response
relationships and poststroke treatments for anti–MMP-9 treatment, the
present data clearly demonstrate that MMP-9 plays a significant
role in brain injury produced by focal stroke.
|
Selected Abbreviations and Acronyms
|
|---|
| BBB |
= |
blood-brain barrier |
| DAB |
= |
3,3'-diaminobenzidine |
| ECM |
= |
extracellular matrix |
| MCA |
= |
middle cerebral artery |
| MCAO |
= |
middle cerebral artery occlusion |
| MMP |
= |
matrix metalloproteinase |
| MMP-1 |
= |
matrix metalloproteinase 1; interstitial
collagenase |
| MMP-2 |
= |
matrix metalloproteinase 2; 72-kD gelatinase |
| MMP-3 |
= |
matrix metalloproteinase 3; stromelysin |
| MMP-9 |
= |
matrix metalloproteinase 9; 92-kD gelatinase |
| SDS-PAGE |
= |
sodium dodecyl sulfate–polyacrylamide gel
electrophoresis |
| TIMP |
= |
tissue inhibitor of metalloproteinase |
|
|
Acknowledgments
|
|---|
The authors are appreciative of Jeffrey S. Culp for his helpful
discussions in conducting this study. The authors are also grateful to
Juanli Gu for her technical assistance.
Received November 21, 1997;
revision received January 14, 1998;
accepted February 3, 1998.
|
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Editorial Comment
Inhibition of Matrix Metalloproteinase-9 Reduces Infarct Size
Valina L. Dawson, PhD, Guest Editor
Department
of Neurology,
Johns Hopkins University School of Medicine,
Baltimore, Maryland
|
Introduction
|
|---|
In the central nervous system inflammatory mediators are
activated in a manner similar to but different from
peripheral inflammatory responses after brain injury or
during neurological disease. The current focus of the research on
inflammatory response in the central nervous system largely consists of
cytokine-induced factors. MMPs are a family of
calcium-dependent, zinc-dependent endopeptidases that
are induced by cytokines. MMPs contribute to the enzymatic
cascade responsible for degradation of ECM proteins such as collagen,
proteoglycan and laminin. In the central nervous system,
endothelial cells, microglia, and astrocytes can
express MMPs in response to injury resulting, in part, in the breakdown
of the BBB and facilitation of leukocyte infiltration and other
inflammatory responses. MMP-2 (gelatinase A) and MMP-9 (gelatinase B)
are the primary MMPs whose levels increase after stroke. Opposing the
actions of metalloproteinases are the TIMPs.
Recent studies indicate that MMP-2, MMP-9, and TIMP increase after
focal ischemia. Thus, these proteins might play critical roles
in the disruption of the BBB and facilitation of leukocyte infiltration
and inflammatory responses following stroke. As reported in the
accompanying article, using an antibody to MMP-9, Romanic and
colleagues observed a decrease in infarct size after focal
ischemic insult in rats, thus identifying MMP-9 as a potential
therapeutic target for the treatment of stroke. In this study the
authors observed MMP-2 and MMP-9 expression in
endothelial cells, neurotrophils, and
macrophages but not in astrocytes or microglia, as other
studies have reported.1 This is likely due to differences
in model systems and mechanism of injury. Romanic and colleagues also
did not observe an increase in TIMP by immunohistochemistry following
ischemic insult, also in contrast to previous
studies.2 The discrepancies between the work described by
Romanic and colleagues and other reports likely reflect the complexity
of the response of MMPs and TIMPs to injury. MMPs and TIMPs are
translated in latent form and then activated by cellular
response to injury. Transcriptional, translational, and
posttranslational regulation of these enzymes is complex, and the
pathways for these events in brain are not known. This is a new and
developing field, and it is anticipated that relatively selective and
specific inhibitors for each MMP will be forthcoming. These
agents might provide significant therapeutic benefit without
deleterious side effects. The current work indicates that selective
modulation of this class of enzymes might have significant positive
benefit for patients suffering from stroke.
|
Selected Abbreviations and Acronyms
|
|---|
| BBB |
= |
blood-brain barrier |
| DAB |
= |
3,3'-diaminobenzidine |
| ECM |
= |
extracellular matrix |
| MCA |
= |
middle cerebral artery |
| MCAO |
= |
middle cerebral artery occlusion |
| MMP |
= |
matrix metalloproteinase |
| MMP-1 |
= |
matrix metalloproteinase 1; interstitial
collagenase |
| MMP-2 |
= |
matrix metalloproteinase 2; 72-kD gelatinase |
| MMP-3 |
= |
matrix metalloproteinase 3; stromelysin |
| MMP-9 |
= |
matrix metalloproteinase 9; 92-kD gelatinase |
| SDS-PAGE |
= |
sodium dodecyl sulfate–polyacrylamide gel
electrophoresis |
| TIMP |
= |
tissue inhibitor of metalloproteinase |
|
Received November 21, 1997;
revision received January 14, 1998;
accepted February 3, 1998.
|
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Abstract
Introduction