From the Departments of Neurology (G.A.R., E.Y.E., J.E.D.), Cell Biology
and Physiology (G.A.R.), and Neuroscience (G.A.R.), University of New Mexico,
Albuquerque, NM.
Correspondence to Gary A. Rosenberg, MD, Department of Neurology, University of New Mexico, Albuquerque, NM 87131. E-mail grosen{at}unm.edu
Methods—Adult rats underwent MCAO for 2 hours by the suture
method. Brain uptake of 14C-sucrose was measured from 3
hours to 14 days after reperfusion. Levels of MMPs and TIMPs were
measured by zymography and reverse zymography, respectively, in
contiguous tissues. Other rats had water and electrolytes measured at
3, 24, or 48 hours after reperfusion. Treatment with a synthetic MMP
inhibitor, BB-1101, on BBB permeability and cerebral edema
was studied.
Results—Brain sucrose uptake increased after 3 and 48 hours of
reperfusion, with maximal opening at 48 hours and return to normal by
14 days. There was a correlation between the levels of gelatinase A at
3 hours and the sucrose uptake (P<0.05). Gelatinase A
(MMP-2) was maximally increased at 5 days, and TIMP-2 was highest at 5
days. Gelatinase B and TIMP-1 were maximally elevated at 48 hours. The
inhibitor of gelatinase B, TIMP-1, was also increased at 48
hours. Treatment with BB-1101 reduced BBB opening at 3 hours and brain
edema at 24 hours, but neither was affected at 48 hours.
Conclusions—The initial opening at 3 hours correlated with
gelatinase A levels and was blocked by a synthetic MMP
inhibitor. The delayed opening, which was associated with
elevated levels of gelatinase B, failed to respond to the MMP
inhibitor, suggesting different mechanisms of injury for
the biphasic BBB injury.
Proteolysis depends on the balance between the proteases and their
inhibitors. Tissue inhibitors to
metalloproteinases (TIMPs), which interfere with the activation and
action of the MMPs, affect the extent and duration of proteolytic
damage.13 Recently, quantitative measurement of
TIMPs was described with the use of a reverse
zymogram.14 Thus, combining measurements of MMPs
by zymography with measurements of the TIMPs with reverse zymography
provides insights into the proteolytic potential for damaging the
extracellular matrix. Synthetic MMP inhibitors, which were
developed for the treatment of cancer and arthritis, have been used to
treat brain disorders.15 16 17 18 19 We hypothesized that
an imbalance between MMPs and TIMPs may increase capillary permeability
in reperfusion injury, contributing to cerebral edema. To test the
hypothesis, MMPs were measured by quantitative zymography and TIMPs by
reverse zymography, and the results were compared with BBB opening and
cerebral edema. An MMP inhibitor was tested for an effect
on BBB permeability and cerebral edema. A preliminary report of this
data has been published.20
BBB Permeability
Quantitative Zymography
Reverse Zymography
Tissue samples were thawed and were mixed 1:1 with nonreducing SDS
loading buffer (New England Biolabs) before loading on gels. Prepared
samples were not boiled or exposed to reducing agents. Prestained
rainbow-colored molecular weight markers (Amersham Life Science) and
HT1080 fibrosarcoma media, which contains TIMP-1 and TIMP-2, were run
in every gel to determine molecular weights of TIMPs. After loading,
gels were electrophoresed at 150 V for 2.5 hours. Following
electrophoresis, all gels were agitated (2x 30 minutes) in 2.5%
Triton X-100 to remove the SDS. Gels were rinsed 3 times in distilled
water, then incubated for 16 hours at 37°C in 50 mL of 50 mmol/L
Tris-HCl (pH 7.6) containing 0.2 mol/L NaCl, 5 mmol/L
CaCl2, 0.02% Brij-35, and 0.02% Azide. Finally,
all gels were stained for 1 hour in Coomassie G-250. Gels were then
destained for approximately 1 hour in 10% acetic acid. Zones of TIMP
activity were seen as dark bands against a clear background.
Zymogram and reverse zymograms were scanned with an Hewlett Packard
ScanJet IIc, and images were analyzed using an image
analysis software program (NIH Image), running on a Macintosh
PowerPC. Image standardization was accomplished using an optical
density step tablet, and the MMP and TIMP activities were measured
using the electrophoretic gel lane calculation option. The initial
series of time points (3 to 24 hours) for TIMP measurements were run
with a gelatinase kindly supplied by Dr Dylan Edwards at the University
of Calgary. The second series of experiments from 48 hours to 14
days were run with a recombinant gelatinase A from Dr William
Stetler-Stevenson. Data was expressed as relative lysis zone (pixels)
divided by protein content in the tissue.
Brain Water and Electrolytes
Statistical Analysis
MMPs and TIMPs
To allow comparison over multiple time points from samples run on
different gels, the amount of gelatinase B in the ischemic side
was normalized by dividing it by the nonischemic side. Optical
density measurements showed a significant increase in gelatinase B
ratios by 48 hours (Figure 3A
Although the mean levels of gelatinases A and B were not increased at 3
hours, plotting the individual values for gelatinase A against sucrose
space revealed a strong correlation at 3 hours (P<0.0003)
and lower significance for gelatinase B (P<0.04) (Figure 4
Ratios of TIMP-1 in the 2 sides showed a statistically significant
elevation at 48 hours that was absent at 5 and 14 days (Figure 3C
Effect of Treatment With a Metalloproteinase Inhibitor
Brain water was significantly reduced by the BB-1101 at 24 hours
(Figure 5B
Matrix-degrading proteases are important in many normal and
pathological processes, including motility of developing cells, spread
of cancer cells, inflammatory responses, and tissue repair after
injury.24 The balance between the proteases and
the inhibitors determines whether there is proteolytic
breakdown of extracellular matrix or inhibition of proteolysis with
buildup of extracellular matrix. Normally, gelatinase A is present
in brain tissue in a latent form, which is activated by a
membrane-bound protease, membrane-type metalloproteinase
(MT-MMP).25 Attachment of TIMP-2 to MT-MMP is
required for the activation MMP-2. Because the activation reaction
proceeds on the membrane surface, proteolysis is spatially constrained.
Gelatinase A is constitutively expressed, making it available for early
tissue injury. Along with the serine protease, plasminogen
activator, MMP-2 is also a critical factor in the
controlled proteolysis during blood vessel regrowth. On the other hand,
gelatinase B is a proinflammatory protease that is released during the
neuroinflammatory response by the stimulation of the cytokines
and immediate early genes. The gelatinase B promoter region contains
both AP-1 and nuclear factor-
Several brain cells produce gelatinases under resting and stimulated
conditions. Astrocytes normally produce latent gelatinase
A.28 29 Microglial cells produce gelatinase B
after stimulation by proinflammatory agents.29 30
Cerebral capillaries produce gelatinases.28
Neutrophils also produce gelatinases, elastase, and cathepsins,
which may contribute to the disruption of the tissue in the secondary
inflammatory response.31 32 33
Intracerebral injection of activated gelatinase
A opened the BBB, and TIMP-2 blocked the gelatinase-induced
opening.10 Tumor necrosis factor-
The initial rise in capillary permeability may have been due to
hyperemia that accompanies the early stages of
reperfusion.36 Treatment with the
hydroxymate-type synthetic inhibitor to MMPs reduced this
initial increase in capillary permeability at 3 hours and modified the
permeability at 48 hours on the nonischemic side. There was a
correlation between the levels of gelatinases at 3 hours and the
increases in the BBB that suggests that they are linked. One possible
explanation for the early effect is that the inhibitor
blocked the activation of the endogenous MMP-2 by MT-MMP.
BB-1101 is a potent inhibitor of MT-MMP (K. Miller,
personal communication, 1998). We have observed by
immunohistochemistry that the MMP-2 is normally found around the
cerebral vessels in astrocytic processes that abut on the capillary
wall and that MT-MMP is also present (S. Mun-Bryce, J.
Wallace, unpublished data, 1998). Another possibility is that
the inhibitor blocked the conversion of tumor necrosis
factor-
A marked increase in TIMP-1 was seen around the time of maximal
rise in the gelatinase B. At 5 days, the BBB was closed, and the levels
of both MMP-9 and TIMP-1 had fallen drastically. TIMP-2 was maximal at
the time of maximal increases in the levels of MMP-2. TIMP-1 inhibits
the action of MMP-9, and TIMP-2 inhibits MMP-2; both have been
implicated in other functions, but the role of TIMPs in brain is
uncertain.
Brain water content rose significantly from the onset of reperfusion,
while the changes in capillary permeability fluctuated. A composite
graph showing the changes in multiple parameters is shown
in Figure 6
We found that the synthetic metalloproteinase inhibitor
prevented the increase in brain water at 24 hours but not at 48 hours.
The initial opening of the BBB was also limited by the
inhibitor, suggesting that it was affecting the initial
increase in capillary permeability. The inability of the
inhibitor to act at 48 hours suggests that the second phase
of injury was related to the neuroinflammatory response, which involves
the infiltrating leukocytes and the endogenous microglial
cells. Free radicals and other proteases, such as elastase, which
are not inhibited by the MMP inhibitor, may be important
because they are released by neutrophils and macrophages and
are disruptive to the capillary.38 Another
possible explanation of the failure of the agent to affect the edema at
48 hours is that the delayed BBB opening is beneficial by helping to
remove fluid from the injury site. Such a mechanism has been proposed
earlier to explain the finding of increased capillary permeability
without cerebral edema after an ischemic
injury.39 Our results suggested that the MMP
inhibitor reduced the capillary permeability at 48 hours,
while the edema was slightly increased, which is consistent
with the notion that the opening of the BBB then may be beneficial, but
further studies are needed to clarify this point.
Our results suggest that the balance between the MMPs and their
inhibitors play a role in reperfusion injury. Inhibition of
the metalloproteinases altered the early damage to the capillary and
controlled the brain edema at 24 hours. However, the failure of the MMP
inhibitors to affect the delayed injury suggest a
multifactorial process. Because of the complex manner in which the
increases and reductions in the proteases and inhibitors
are woven together during the reperfusion process, attempts at altering
the patterns with drugs will be complicated.
Received January 7, 1998;
revision received May 18, 1998;
accepted June 1, 1998.
Department
of Neurology,
Washington University School of Medicine,
St Louis, Missouri
Dr Rosenberg's laboratory was the first to apply zymograms and reverse
zymograms to determine changes in the activities of MMPs and their
inhibitors in the rat stroke model. This study, together
with earlier works by the same group, provide interesting findings on
the activation of a complex biochemical cascade that may contribute to
ischemic brain edema.
It should also be noted that in the present study, the correlation
of MMP activities and BBB breakdown, based on sucrose space and brain
water content, was not as tight as expected. This may be partly caused
by the sampling method applied to this study. Lack of BB-1101 effects
on the ultimate outcomes at 48 hours, especially on the brain water
content, suggests the need to measure MMP activities following BB-1101
treatment to possibly maximize the intended therapeutic effects.
In summary, the Rosenberg group pioneered in the demonstration of an
activated MMP pathway in the setting of cerebral
ischemia with or without reperfusion. MMP expression may
contribute to the postischemic BBB breakdown after
ischemic stroke. However, additional studies are needed before
the roles of MMPs in the pathogenesis of ischemic brain edema
can be fully defined.
Received January 7, 1998;
revision received May 18, 1998;
accepted June 1, 1998.
© 1998 American Heart Association, Inc.
Original Contributions
Matrix Metalloproteinases and TIMPs Are Associated With Blood-Brain Barrier Opening After Reperfusion in Rat Brain
Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
Background and
Purpose—Reperfusion disrupts cerebral capillaries, causing
cerebral edema and hemorrhage. Middle cerebral artery occlusion
(MCAO) induces the matrix-degrading metalloproteinases, but their role
in capillary injury after reperfusion is unknown. Matrix
metalloproteinases (MMPs) and tissue inhibitors to
metalloproteinases (TIMPs) modulate capillary permeability. Therefore,
we measured blood-brain barrier (BBB) permeability, brain water and
electrolytes, MMPs, and TIMPs at multiple times after
reperfusion.
Key Words: blood-brain barrier • brain edema • cerebral ischemia • reperfusion injury • matrix metalloproteinases • type IV collagenases
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
Reperfusion of ischemic tissue with
thrombolytic agents shortly after the infarct reduces
ischemic damage.1 However, a delay in
treatment increases the risk of hemorrhagic transformation and cerebral
edema.2 3 Several mechanisms have been proposed
to explain the tissue damage associated with reperfusion of
ischemic tissue. Reintroduction of oxygenated blood
into the damaged region enhances the production of free
radicals, recruits neutrophils and macrophages, and releases
proteases. Reperfusion causes a biphasic opening of the blood-brain
barrier (BBB).4 5 Free radicals,
cytokines, and proteases may mediate the attack on the
capillary.6 7 8 9 Matrix metalloproteinases (MMPs)
attack the basal lamina around cerebral capillaries, which contains
type IV collagen, fibronectin, laminin, and heparan sulfate. Direct
intracerebral injection of one of the MMPs, gelatinase
A (72-kDa type IV collagenase or MMP-2), caused an increase
in capillary permeability and
hemorrhage.10 In rats with permanent middle cerebral artery occlusion (MCAO), a large
increase in the MMP gelatinase B (92-kDa type IV
collagenase or MMP-9) occurred 12 to 24 hours after the
injury,11 when capillary permeability was
elevated.12 Thus, the MMPs may be involved in the
increased capillary permeability seen in reperfusion injury.
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
MCAO With Reperfusion
The study was approved by the University of New Mexico Animal
Care Committee and conformed to the National Institutes of Health
Guidelines for use of animals in research. Adult Wistar rats, weighing
280 to 320 g, were anesthetized with 1.5% halothane in
70% nitrous oxide and 30% oxygen. Temporary MCAO was done by
insertion of an intraluminal nylon suture with a bulb on the
end.21 Neck vessels were exposed through a
midline incision, and branches of the right external carotid artery
were isolated and ligated. A 6-0 silk suture was loosely tied around
the external carotid artery stump. A 4-0 monofilament nylon suture was
introduced into the external carotid and advanced into the MCA. A silk
suture around the stump was tied down onto the thread with the end of
the thread protruding slightly. Reperfusion was achieved by slowly
pulling the thread back.
Brain uptake of sucrose was measured in 55 rats that had
undergone 2 hours of MCAO with reperfusion for 3, 6, 15, 24, 48, 120,
or 336 hours. BBB permeability was measured by a modification of the
brain uptake method.10 22 Ten minutes before
death, the rats were anesthetized with 1.5% halothane in 70%
N2O and 30% O2 and infused
intravenously over 30 seconds with 10 µCi of
14C-sucrose (Dupont/New England Nuclear).
Samples of blood were drawn from the femoral vein, and the heart was
stopped with an intracardiac injection of a saturated potassium
chloride solution. The brain was removed and frozen in 2-methylbutane
cooled to -80°C. A 5-mm tissue section was removed from a region
approximately 5 mm away from the tip of the frontal lobe, which is
the main site of infarction. A piece of tissue, which contained cortex
and caudate from the region of the infarct, was obtained from a 5-mm
coronal section. A section was similarly taken from the noninfarcted
hemisphere. For BBB, measurements tissue was dissolved in Aquasol (New
England Nuclear), and brain and blood samples were counted for
radioactivity in a liquid scintillation counter. The ratio of brain
sucrose content to that in the blood was calculated. For zymography,
tissues are taken from a contiguous 5-mm coronal section directly
posterior to the one used for BBB measurements. Similar regions of the
infarcted and noninfarcted sides were studied.
The zymographic method for measurements of MMPs in rat brain has
been described previously.23 Briefly, tissue
samples were weighed and dissolved in Triton X-100. The tissue was
centrifuged and the supernatant removed for zymography in SDS
gels with gelatin. Electrophoresis was run at 150 V for 1 hour. The
sample gels were placed in Triton X-100 for 30 minutes to remove the
SDS and restore the proteins to activity. After incubation, the gels
were stained with Coomassie G-250 dye. The molecular standards and
HT1080 conditioned media, which contains gelatinases A and B, were used
to calibrate the molecular weights. Protein content in the samples was
measured by the BCA method with a kit (Pierce Co).
Reverse zymography was performed as recently
described.14 Polyacrylamide minigels
(15%) are prepared: 5.0 mL Protogel (30% ultrapure, National
Diagnostics); 2.5 mL of 1.5 mol/L Tris-HCl, pH 8.8; 1.67 mL
porcine gelatin at 15 mg/mL; 0.83 mL distilled water; 0.2 mL of 10%
SDS; 6.4 µL of purified gelatinase A (0.252 µg/µL; a gift from Dr
W.G. Stetler-Stevenson at the National Cancer Institute); 50
µL of 10% ammonium persulfate; and 5 µL of
N,N,N',N'-tetra-methylethylenediamine
(TEMED). Gels were allowed to polymerize for 1.5 to 2 hours before the
stacking gel was added (5.6 mL distilled water; 4.15 mL 0.5 mol/L Tris,
pH 6.8; 1.66 mL Protogel 30%; 125 µL 10% SDS; 10 µL TEMED; and
200 µL 10% APS). The stacker was allowed to polymerize for 1 hour
before samples were loaded.
Fifty-two rats had brain water and electrolyte content measured
after 2 hours of MCAO followed by 3, 24, or 48 hours of reperfusion
with or without treatment with MMP inhibitor. Brain water
was measured in tissue with the wet/dry method. Tissue was frozen, and
sections were taken from the ischemic and nonischemic
hemispheres. The tissue was weighed wet and dried in a 100°C oven for
48 hours and reweighed. After drying, it was extracted for electrolyte
measurement with a flame photometer (Corning Corp). Rats with
temporary MCAO (tMCAO) for 2 hours were treated with a
metalloproteinase inhibitor, BB-1101 (British
Biotechnology), after either 3, 24, or 48 hours of reperfusion. The
BB-1101 (30 mg/kg) was given intraperitoneally at
the onset of the ischemia and repeated after 2 hours of
reperfusion in the 24-hour and 48-hour studies. For the BBB studies,
treated rats underwent 2 hours of tMCAO followed by 3, 24, or 48 hours
of reperfusion, and they were compared with untreated rats from the BBB
series. For the edema and electrolyte studies, treated rats underwent
2-hour tMCAO followed by 3, 24, or 48 hours of reperfusion, and they
were compared with untreated rats. The effect of the agent on
physiological parameters was assessed
in 5 rats. Blood glucose, mean arterial blood pressure, and
temperature were measured before injection of BB-1101, which was given
10 minutes after the start of the experiment and at 4 and 24 hours
later.
BBB data was tested for statistical significance with an ANOVA,
using the Bonferroni correction for multiple t tests (Prizm,
GraphPad). The zymographic data was normalized by dividing the
ischemic hemisphere by the nonischemic one. An ANOVA
was run on the normalized data to determine significantly different
time points with the Newman-Keuls correction for multiple comparisons.
Significance was taken as a probability of P<0.05. The
results were expressed as mean±SEM.
Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
BBB Permeability and Brain Water and Electrolytes
Uptake of sucrose into the brain was significantly increased after
3 hours of reperfusion and was maximal at 48 hours (Figure 1
). By 3 hours of reperfusion, the brain
water was increased, reaching maximal levels by 24 and 48 hours, with
sodium and potassium paralleling the changes in water content (Table 1). Significant increases in water
content were found at 3, 24, and 48 hours. Sodium was significantly
increased in the ischemic side compared with sham-operated
controls at 24 and 48 hours, whereas the sodium was increased on the
nonischemic side at 48 hours. Potassium was significantly
decreased at 24 hours and increased on the nonischemic side at
48 hours.
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Figure 1. Uptake of 14C-sucrose into brain in
rats with temporary occlusion of the MCA by the suture method. The
artery was occluded with an intraluminal thread for 2 hours and then
withdrawn to reperfuse the brain. Adult rats were injected with
14C-sucrose at the end of the reperfusion period
and killed 10 minutes later. Samples from brain and blood were
collected, and brain sucrose uptake was calculated as a percentage of
sucrose in brain to that in blood (Sucrose Space %). The sucrose
spaces in the ischemic sides were compared by ANOVA with
Bonferroni corrections. A significant increase in the uptake of sucrose
occurred at 3 and 48 hours compared with all other times.
*Statistically significant increases with P<0.05.
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Table 1. Water and Electrolytes in Cerebral Tissue After
2-Hour MCAO With Various Times of
Reperfusion
Representative zymograms and reverse zymograms
from reperfused tissues are shown in Figure 2. Elevated levels of gelatinase B at
92-kDa are seen in the reperfused tissue at 48 hours (Figure 2A).
Normally, MMP-9 is undetectable in rat brain.11
All tissue samples showed bands at 72 kDa from MMP-2, which is
constitutively expressed. Reverse zymograms showed dark bands at 28 kDa
and 21 kDa from TIMP-1 and TIMP-2, respectively (Figure 2B).
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Figure 2. Representative zymogram and
reverse zymograms. The samples are from the 48-hour to 14-day series.
A, Gelatin-substrate zymogram with molecular weights as shown on the
side determined from HT1080 run in a lane that is not shown. Lane 1 is
from nonischemic (NI) tissue from the caudate after 2-hour MCAO
and 48-hour reperfusion. Lane 2 is the same except the tissue is from
the ischemic side (I). Lanes 3 through 6 are 120 hours and 336
hours of reperfusion as labeled. B, Reverse zymogram from brain
tissue at the same time points as in A. Molecular weights are derived
from TIMP standards and HT1080. The higher band at 28 kDa is from
TIMP-1 and possibly a glycosylated form of TIMP-3, while the lower
21-kDa band contains TIMP-2. The massive increase in TIMP-1 is seen at
48 hours in the reverse zymogram, corresponding to the increase in
gelatinase B at the same time in the zymogram. ). The
increase in gelatinase B began by 15 hours but was not significant at
that time. After 48 hours, there was a drastic reduction in the levels
of gelatinase B. Gelatinase A ratios were maximal at 5 days (Figure 3B).
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Figure 3. Effect of temporary MCAO on production of
gelatinases in tissue from the region of the infarct (ischemic)
and the corresponding site in the uninjured side (nonischemic).
Rats had temporary MCAO for 2 hours with an intraluminal thread and
reperfusion for times shown. Quantitative zymograms were prepared with
brain tissue from the ischemic and nonischemic
hemispheres. The levels of gelatinases are given as ratios of the
ischemic (i) to the nonischemic (ni) side. A,
Ratio of gelatinase B at different times after reperfusion. The
differences between the 48-hour times and all others except 15 and 24
hours were significantly different, suggesting that an increase in
gelatinase B was beginning at 15 hours. B, Ratios of gelatinase A were
similar at all times except 120 hours after reperfusion. C, Ratios of
TIMP-1 showed a single massive increase at 48 hours. D, Ratios of
TIMP-2 were highest 120 hours, which was not different from 15, 48, or
336 hours. Statistical significance calculated by an ANOVA with
Newman-Keuls correction for multiple comparisons:
+P<0.05; *P<0.01;
**P<0.001. ). No correlation was found at 24 hours
for either of the enzymes (data not shown). The marked increase in
gelatinase B at 48 hours coincided with the maximal increase in sucrose
uptake.
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Figure 4. Correlation of the levels of gelatinases with
sucrose spaces for individual animals. Values are shown for rats with 3
hours of reperfusion. 
indicates the gelatinase A (Gel A); 
,
gelatinase B (Gel B). A highly significant correlation between the
values of gelatinase A and sucrose space was seen at 3 hours (F value
of 21.5 with P<0.0003). For gelatinase B, the
significance was less (F value of 4.8 with
P<0.04).
).
Ratios of TIMP-2 were more erratic, with maximal levels seen at 5 days,
reaching significance compared with the 3-, 6-, and 24-hour values.
Maximal ratios were seen at 5 days when the ratios for gelatinase A
were also increased (Figure 3D).
The synthetic metalloproteinase inhibitor BB-1101 did
not affect blood glucose level, mean arterial blood
pressure, or temperature for the 24 hours during which these
parameters were measured (Table 2). BB-1101 significantly decreased
uptake of sucrose at 3 hours in the ischemic hemisphere and at
48 hours in the nonischemic hemisphere (Figure 5A). It also lowered the sucrose space at
48 hours in the ischemic side, but the results did not achieve
statistical significance.
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Table 2. Effect of BB-1101* on Blood Glucose, Temperature,
and Mean Arterial Blood Pressure at 0, 4, and 24 Hours After
Injection
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Figure 5. A, Effect of treatment
with BB-1101 (MMPI) on the sucrose space. Ischemic (ISC) and
nonischemic (NISC) sides are given. The numbers under the bars
refer to occlusion/reperfusion times. Treated rats had a significant
reduction in the sucrose space at 3 hours in the ischemic
hemisphere. The reduction in the ischemic hemisphere failed to
reach statistical significance at 48 hours but was significant on the
nonischemic side. B, Water content was measured by weighing
tissue wet and after it was dried overnight in an oven.
Treatment with a metalloproteinase inhibitor (BB-1101)
significantly decreased edema in the ischemic hemisphere of
treated rats compared with untreated rats at 24 hours but had no effect
at 48 hours. *Significant differences. ). No effect of the agent was seen at 48 hours, however.
Administration of the drug after either a 1- or 2-hour delay failed to
affect the cerebral edema at 24 or 48 hours, suggesting that the agent
was acting to alter an early event (data not shown).
Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
Reperfusion for 3 and 48 hours after 2-hour occlusion of the MCA
opened the BBB, with the maximal opening at 48 hours. The initial
opening corresponded with increased levels of gelatinase A, whereas the
second one occurred when gelatinase B was markedly increased. Maximal
levels of gelatinase A were seen at 5 days after reperfusion, during
the time when the repair process had begun. An inhibitor to
metalloproteinases blocked the initial opening of the BBB and also the
edema at 24 hours, suggesting that they were related to a
metalloproteinase. Tissue inhibitor to metalloproteinase-1
was markedly increased at 48 hours, and TIMP-2 was maximally increased
at 5 days. These results show that MMPs and TIMPs are dramatically
affected by reperfusion but contribute in a complex manner to
reperfusion injury. 
B sites, which respond to a wide
variety of proinflammatory stimuli. Released in a proform, gelatinase B
requires an activation step, which has not been reported for the in
vivo situation, but may involve stromelysin, other proteases, or free
radicals.26 27 
, which is
formed in ischemic tissue, increased gelatinase B at 24 hours
when the BBB was maximally opened, and an inhibitor to
metalloproteinases, Batimistat, reduced the capillary
injury.18 Cerebral capillaries are surrounded by
a basal lamina composed of type IV collagen, fibronectin, and laminin,
which are attacked by the proteases, including
gelatinases.34 Ischemia/reperfusion
injury in monkeys causes loss of laminin in the basal lamina, which
adds evidence to the concept that proteolytic disruption of the basal
lamina contributes to the BBB injury.35 from a latent to an active form, preventing it from damaging
the capillary.37 The delayed opening of the BBB
seen at 48 hours was associated with a large increase in gelatinase B,
which may have been endogenous from astrocytes and
microglia or exogenous from the invading neutrophils and
macrophages. However, the MMP inhibitor failed to
interfere with the BBB damage or edema at that time. . At 24 hours when the brain
water was high, the sucrose space had returned to normal, suggesting
that the cytotoxic component of the brain edema predominated. Sodium
content was high and potassium low at that time, as expected for
cytotoxic edema. However, by 48 hours the water content remained high
with elevated levels of sodium and potassium. Furthermore, the sucrose
space was maximal. Thus, vasogenic edema seemed to also contribute to
the water changes at the later time.
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Figure 6. Composite figure showing the relationship and time
course of the brain edema, sucrose (Suc) space, and gelatinases. The
data is from the earlier figures showing the individual results. Water
content is increased by 3 hours and continues to steadily rise.
Capillary permeability shows a biphasic response with peaks at 3 and 48
hours. Gelatinase B peaks at 48 hours, while gelatinase A is increased
maximally at 5 days. Geli/Gelni
indicates gelatinases as ratio of ischemic (i) to
nonischemic (ni) side.
Acknowledgments
These studies were supported by grants from the National
Institutes of Health (RO1-NS21169-10) to Dr Rosenberg. The synthetic
metalloproteinase inhibitor was a gift from Dr A. Gearing
of British Biotechnology.
Footnotes
Presented in part at the meeting of the Society of Cerebral Blood Flow and Metabolism, Baltimore, Md, June 16, 1997; published in the Proceedings of the Princeton Conference, Memphis, Tenn, March 22, 1996.
References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
precursor by
metalloproteinases. Nature. 1994;370:555–557.[Medline]
[Order article via Infotrieve]
Editorial Comment
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
Breakdown of blood-brain barrier (BBB) after cerebral
ischemia–reperfusion may involve a multitude of molecular
events, including the engagement of such mediators as free radicals,
cytokines, and others. In the preceding article by Rosenberg
and colleagues, a time-dependent correlation of
postischemic BBB breakdown and the expression of matrix
metalloproteinases (MMPs) was noted. An increase in sucrose space
reflecting the extent of BBB breakdown was correlated with gelatinase A
expression at 3 hours after ischemia. The enhanced proteolytic
activity, suggesting a perturbed balance of proteases and their
inhibitors, may contribute to the postischemic
opening of the BBB. Rosenberg and associates also found an increase in
tissue MMP inhibitor activity, especially at later time
points. It is interesting to note that the early BBB opening, which was
presumably related to early gelatinase A expression, was reduced by a
synthetic MMP inhibitor, BB-1101. However, the second phase
of postischemic BBB breakdown and the ultimate outcome was
not affected by treatment with this protease inhibitor.
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