|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 569-576
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Neuroserpin reduces cerebral infarct volume and protects neurons
from ischemia-induced apoptosis
Manuel Yepes,
Maria Sandkvist,
Mike K. K. Wong,
Timothy A. Coleman,
Elizabeth Smith,
Stanley L. Cohan, and
Daniel A. Lawrence
Department of Biochemistry, American Red Cross Holland Laboratory,
Rockville, MD; Department of Protein Development, Human Genome Sciences
Inc, Rockville, MD; Department of Neurology, Georgetown University
Medical Center, Washington, DC.
 |
Abstract |
Neuroserpin, a recently identified inhibitor of tissue-type
plasminogen activator (tPA), is primarily localized to neurons within
the central nervous system, where it is thought to regulate tPA
activity. In the present study neuroserpin expression and its potential
therapeutic benefits were examined in a rat model of stroke.
Neuroserpin expression increased in neurons surrounding the ischemic
core (ischemic penumbra) within 6 hours of occlusion of the middle
cerebral artery and remained elevated during the first week after the
ischemic insult. Injection of neuroserpin directly into the brain
immediately after infarct reduced stroke volume by 64% at 72 hours
compared with control animals. In untreated animals both tPA and
urokinase-type plasminogen activator (uPA) activity was significantly
increased within the region of infarct by 6 hours after reperfusion.
Activity of tPA then decreased to control levels by 72 hours, whereas
uPA activity continued to rise and was dramatically increased by 72 hours. Both tPA and uPA activity were significantly reduced in
neuroserpin-treated animals. Immunohistochemical staining of basement
membrane laminin with a monoclonal antibody directed toward a cryptic
epitope suggested that proteolysis of the basement membrane occurred as
early as 10 minutes after reperfusion and that intracerebral
administration of neuroserpin significantly reduced this proteolysis.
Neuroserpin also decreased apoptotic cell counts in the ischemic
penumbra by more than 50%. Thus, neuroserpin may be a naturally
occurring neuroprotective proteinase inhibitor, whose therapeutic
administration decreases stroke volume most likely by inhibiting
proteinase activity and subsequent apoptosis associated with focal
cerebral ischemia/reperfusion.
(Blood. 2000;96:569-576)
© 2000 by The American Society of Hematology.
| |
Introduction |
Stroke is the second most common cause of death in the
world after heart disease and a leading cause of
disability.1 It is estimated that in the United States
someone suffers a stroke about every minute and a person dies of stroke
about every 3.5 minutes.2 Various strategies have been used
to reduce stroke morbidity and mortality, one of which has been
thrombolysis with tissue-type plasminogen activator (tPA) to restore
cerebral blood flow to ischemic brain tissue. Thrombolysis with tPA
produces arterial recanalization in 40% to 67% of
patients3 and is associated with absolute improvement in
neurologic function after 90 days in 12% of the patients treated
within 3 hours of the onset of symptoms.4 However, in
seeming contradiction to these results, animal studies have
demonstrated that tPA-deficient mice have a 41% decrease in stroke
size and a 61% increase in neuronal survival compared with wild-type
animals following occlusion of the middle cerebral artery
(MCA).5 Although these results have been recently challenged,6,7 they have also been reproduced by
others.8 Furthermore, this latter study also demonstrated
that animals deficient in plasminogen had an increase in stroke volume,
whereas animals deficient in the primary plasmin inhibitor,
 2-antiplasmin, had a decrease in stroke size similar to
tPA null mice, suggesting a plasminogen-independent function for tPA in
cerebral ischemia.8
In both rats and mice, tPA expression is increased by events that
require neuronal plasticity, such as synaptic remodeling, long-term
potentiation, kindling, and seizures.9-11 Expression of tPA
is also correlated with central nervous system (CNS) development and
maintenance, and in the modulation of cell-cell and cell-extracellular matrix (ECM) interactions.12-14 As in stroke, tPA-deficient
mice are protected from excitotoxin-induced neuronal death. However, in
contrast to stroke, plasminogen-deficient mice are also protected from
excitotoxic injury,15-17 and it has been suggested that tPA and plasminogen may promote excitotoxin-induced neuronal death through
proteolysis of the neuronal ECM.18
Following stroke there is a densely ischemic area where neurons are
irreversibly damaged, surrounded by an area known as "ischemic penumbra," where cerebral blood flow is sufficiently decreased to
abolish electrical potentials yet sufficient to allow maintenance of
membrane potentials and cellular ionic homeostasis.19-21
This zone of penumbra has also been observed in magnetic resonance image studies of rats in the area surrounding the necrotic
core.22-24 With time, this potentially salvageable area of
penumbra, or reversible ischemia, tends to become infarcted. In vivo
microdialysis has demonstrated that after cerebral ischemia there is a
large release of excitotoxins 25-28 not only in the
infarcted core but also in the area of ischemic penumbra,29
where the presence of apoptotic cells has also been
described.30-32 Because tPA may play an significant role in
both excitotoxin- and ischemia-induced neuronal
degeneration,5,8,15-17,33 it is possible that an inhibitor
of tPA might play an important role in neuronal survival after stroke.
Neuroserpin is a recently identified member of the serine proteinase
inhibitor (serpin) gene family34,35 that reacts
preferentially with tPA, having a second-order rate constant for the
inhibition of tPA of 6.2 × 105
M 1s1.36
Furthermore, neuroserpin expression is primarily localized to regions of the brain where tPA has been previously
identified.36 The present study demonstrates that
neuroserpin is expressed in the area of ischemic penumbra in an animal
model of focal cerebral ischemia/reperfusion. Moreover, intracerebral
administration of neuroserpin after stroke decreases stroke volume,
reduces basement membrane proteolysis, and diminishes the number of
cells with apoptotic features in the area of ischemic penumbra. Thus,
the data presented suggest that neuroserpin, a selective, naturally occurring inhibitor of tPA, may play an important role in neuronal survival after stroke.
| |
Materials and methods |
Animal preparation and surgery
Adult male Sprague-Dawley rats weighting 350 to 400 g were used.
Anesthesia was induced with 4% halothane, 70% nitrous oxide, and a
balance of oxygen and was maintained with 2% halothane and 70%
nitrous oxide during the surgical procedure. Rats were intubated endotracheally and mechanically ventilated. Arterial blood pressure and
blood gases were monitored. Body temperature was maintained at
37.5 ± 0.5°C with a warming blanket (Animal Blanket Control Unit, Harvard Apparatus) and controlled with a rectal thermistor and a
probe inserted into the masseter muscle. The MCA was exposed and
cauterized with a microbipolar coagulator (Non-Stick Bipolar Coagulation Forceps, Kirwan Surgical Products, Marshfield, MA) above
its crossing point with the inferior cerebral vein as described elsewhere.37 Animals were then placed on a stereotactic
frame and 20 µL of either 30 µmol/L active neuroserpin in
phosphate-buffered saline (PBS), 30 µmol/L inactive elastase-cleaved
neuroserpin in PBS, or 20 µL of PBS only was injected intracortically
with a Hamilton Syringe through the burr hole. Comparison of untreated animals (no injection) to PBS-treated rats revealed no significant difference in stroke volume, indicating that the injection itself did
not contribute to the infarct size (data not shown). After the
intracortical injections, the left common carotid artery was exposed
through a midline cervical incision and temporarily occluded for 1 hour
with a microaneurysm clip (8 mm, 100 g pressure; Roboz Surgical
Instruments, Rockville, MD).38 Animals were then allowed to
recover under the heating lamp, returned to their cages, and given free
access to water.
Infarct volume
Rats were anesthetized with pentobarbital intraperitoneally 72 hours
after infarction and brains were removed after transcardiac perfusion
with PBS and paraformaldehyde 4% (Fisher Scientific, HC-200). The
entire brain was embedded in paraffin and coronal sections, 20 µm
thick, were cut through the rostrocaudal extent of the brain (Figure
1). The sections were stained with
hematoxylin-eosin and using the NIH Image Analyzer System, the total
volume of each infarction was determined by the integration of the
areas of 8 chosen sections and the distances between them. The rostral
and caudal limits for the integration were set at the frontal and occipital poles of the cortex.39 Statistical significance
between groups of animals was identified by a Student t test.
View larger version (80K):
[in this window]
[in a new window]
| Fig 1.
Rat brain sections 72 hours after reperfusion
. Hematoxylin-eosin stain of 3 representative sections from the same
brain 72 hours after reperfusion. The infarcted area is indicated with
arrows, and the box indicates the location where higher resolution
analysis was performed (magnification × 5).
|
|
TUNEL staining
Paraffin-embedded sections (5 µm) from neuroserpin- and
control-treated animals killed at 6, 24, 48, and 72 hours after
reperfusion were examined for TUNEL reactivity using the Apoptag Kit
(Oncor, Gaithesburg, MD). Paraffin sections were dewaxed, rehydrated, and treated with proteinase K (20 µg/mL) and blocked for endogenous peroxidase activity with 3% H2O2. Subsequent
end-labeling was done with TdT enzyme at 37°C for 1 hour.
Anti-digoxigenin peroxidase conjugate was applied to the tissue for 30 minutes at room temperature. The slides were developed with peroxidase
substrate DAB for 5 minutes (Sigma, St Louis, MO), washed in
dH2O for 5 minutes, and counterstained with 0.5% methyl
green for 10 minutes. To quantitate the presence of cells with
apoptotic bodies, an area surrounding the ischemic core extending from
the cerebral cortex to the most anterior (septal) part of the
hippocampus was imaged in neuroserpin- and control-treated animals.
Histologic features used by light microscopy to identify apoptosis
depended on recognition of dark-brown rounded or oval apoptotic
bodies.40,41 Statistical significance between groups of
animals was identified by a Student t test.
Zymography
For sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) zymography the region containing the stroke in brains from
neuroserpin- and PBS-treated animals killed at 6 and 72 hours after
reperfusion were dissected and slices of approximately 600 mg were
frozen in dry ice and stored at  70°C. A similar portion of brain
was dissected from the same area in the contralateral hemisphere in
both neuroserpin-treated and control animals. Protein extracts were
prepared in 1.2 mL extraction buffer as described.36 The
protein concentration was then determined, and 30 µg of extract (approximately 1 µL) was mixed with nonreducing sample buffer and
subjected to SDS-PAGE on a 10% gel (Novex, San Diego, CA). Human tPA
0.3 ng (Genentech, San Francisco, CA) and a rat kidney extract
containing urokinase-type plasminogen activator (uPA) prepared in the
same way as the brain extracts were included as positive controls and
the identity of each PA was determined by including either anti-tPA or
anti-uPA in the indicator film (data not shown). Following
electrophoresis, the gel was soaked in 2.5% Triton X-100 for
2 × 45 minutes to remove the SDS. An indicator gel was prepared by
mixing 1.25 mL of an 8% solution of boiled and centrifuged milk in
PBS, 5 mL PBS, and 3.75 mL of a 2.5% agar solution prewarmed at
50°C. Plasminogen (Molecular Innovations, Royal Oak, MI), was added
to a final concentration of 30 µg/mL and the solution mixed and
poured onto a prewarmed glass plate. The Triton X-100 soaked gel was
applied to the surface of the plasminogen-milk indicator gel and
incubated in a humid chamber at 37°C. Milk indicator gel without
plasminogen was also included as a control. The relative increase of
tPA and uPA ipsilateral to the stroke at 6 hours after reperfusion was
quantified by scanning a photograph of the SDS-PAGE zymography gel
taken at an early time of development, before full lysis had occurred,
and using the NIH Image Analyzer System. Normal baseline PA activities
were calculated from the average of the activity present in 6 independent contralateral samples for which the coefficient of
variation was less than 0.2. Control analysis of purified tPA by this
method demonstrated that lysis was linear over at least an 8-fold range with a correlation coefficient (r) of 0.994. Statistical significance between groups was identified by a Student t test.
For the in situ proteinase activity assay, brains from neuroserpin- and
control-treated animals killed at 6 and 72 hours after reperfusion
(n = 3 for each condition at each time point) were frozen in optimal
cutting temperature compound (OCT) and stored at
70°C. Cryostat sections 8 µm thick were examined for PA
activity in overlays prepared as described.14 The overlay
mixture (150 µL) was applied to prewarmed tissue sections and spread
under glass coverslips. Slides were incubated in a humid chamber at 37°C and developed. Control sections were overlaid with a milk agar
mixture without plasminogen. Other controls included those in which
either 100 µg/mL anti-tPA (a generous gift of Tom Podor, MacMaster
University) or anti-uPA (Chemicon International, Temecula, CA)
antibodies or 5 µmol/L neuroserpin36 were included in
addition to plasminogen.
Immunohistochemistry
All immunohistochemistry was performed on 5-µm
deparaffinized-embedded sections. The sections were first immersed in
methanol 0.3% H2O2 for 30 minutes and then
either preincubated directly with 10% serum (either horse or goat) or
first treated with 0.04% pepsin in 0.1 N HCl for 20 minutes at
23°C before being blocked with serum. All sections were also
developed with the ABC reagent (Vector Laboratories, Burlingame, CA),
using the DAB chromogen for 4 minutes, after which the sections were
counterstained with Mayer's hematoxylin for 1 minute. For neuroserpin
staining, adult male Sprague-Dawley rats that were not injected with
neuroserpin or PBS were killed 6, 24, 48, 72, 96, or 168 hours after
bipolar coagulation of the MCA, or sham operation, and sections were
prepared as above and stained with rabbit antihuman neuroserpin as
described.36 For tPA, uPA, and laminin, both control and
neuroserpin-treated animals were examined. For tPA, the sections were
stained with affinity purified sheep antihuman tPA (a generous gift
from Tom Podor, MacMaster University), at 1:800 dilution after pepsin
digestion. For uPA goat antihuman, uPA (Chemicon International-AB767)
was used at 1:200 dilution after pepsin digestion. For laminin
staining, a murine monoclonal antihuman laminin (Chemicon
International-MAB2920) was used at a 1:4000 dilution either with or
without pepsin digestion as above. For all immunohistochemical
analysis, n  2 for each condition at each time point except for
neuroserpin staining at 96 and 168 hours, for which n = 1 each.
| |
Results |
Neuroserpin expression after stroke
Because tPA may contribute to neuronal death after cerebral
infarction, increased expression of neuroserpin might play an important
role in neuronal survival after stroke. To examine the expression of
neuroserpin after cerebral ischemia, immunohistochemical staining of
brain sections was performed at 6, 24, 48, 72, 96, and 168 hours after
MCA occlusion and reperfusion. Figure 1 shows 3 representative brain
sections harvested 72 hours after reperfusion and stained with
hematoxylin-eosin. The infarct is clearly evident as the lighter
stained tissue in the cortex of the left hemisphere, and the box
indicates the area where higher resolution analysis was performed.
Neuroserpin immunoreactivity was increased in the area surrounding the
ischemic core (penumbra) and in the ipsilateral hippocampus as early as
6 hours after stroke and remained elevated up to 168 hours when
compared with the contralateral, nonischemic hemisphere or with
sham-operated controls (data not shown). The peak of neuroserpin
immunoreactivity in both the number of neuroserpin-positive cells and
in the intensity of the staining appeared to be at 48 hours following
reperfusion (Figure 2). The apparent rapid
increase in neuroserpin expression following infarction suggests that
the surrounding surviving cells may be up-regulating neuroserpin
expression in response to the ischemic insult.
View larger version (153K):
[in this window]
[in a new window]
| Fig 2.
Immunohistochemical staining of neuroserpin in brain 48 hours after reperfusion.
Panel A shows the area of penumbra, panel B shows a similar area of the
cortex contralateral to the stroke and panels C and D show the
hippocampus. Panels A and C are ipsilateral to the stroke and panels B
and D are contralateral (magnification × 100 in A and B and
× 40 in C and D).
|
|
Effect of neuroserpin on stroke volume
To see if neuroserpin could reduce neuronal cell death after stroke
with subsequent preservation of normal brain tissue, neuroserpin was
administered intracerebrally immediately following MCA occlusion. Comparison of stroke volume between control and neuroserpin-treated animals 72 hours after reperfusion indicated that intracortical injection of 30 µmol/L neuroserpin reduced stroke size by 64%, from
161 mm3 in control animals to 58 mm3 in
neuroserpin-treated animals (Figure 3). In
contrast, stroke volume in animals treated with inactive neuroserpin,
cleaved in its reactive center loop, showed no decrease in stroke size
relative to control animals, suggesting that active neuroserpin is
required to reduce stroke volume (Figure 3).
View larger version (21K):
[in this window]
[in a new window]
| Fig 3.
Quantitative analysis of infarct volume 72 hours after
reperfusion.
Quantitation of the stroke volume was performed as described in
"Materials and methods." PBS indicates animals injected with PBS
(n = 8); Ns, animals injected with neuroserpin (n = 8); Cl-Ns,
animals injected with elastase-cleaved inactive neuroserpin (n = 2).
P values relative to the PBS-treated animals < .01 are shown,
and errors represent SEM.
|
|
Proteinase activity after stroke
The fact that only the active inhibitory form of neuroserpin reduced
stroke volume suggests that neuroserpin acts primarily by blocking
proteinase activity, possibly tPA activity.36 To examine
proteinase activity following stroke, and to determine the effect of
neuroserpin treatment on proteinase activity, 2 different assays were
used. The first, SDS-PAGE zymography was performed on extracts of
tissues dissected from the cortex, either ipsilateral or contralateral
to the stroke of both PBS- and neuroserpin-treated animals (Figure
4A). After electrophoresis and removal of
SDS, the gels were overlaid onto milk-agarose gels with or without plasminogen. In the absence of plasminogen no proteinase activity could
be detected in any of the extracts, whereas addition of plasminogen to
the milk-agarose mixture demonstrated that both tPA and uPA activity
were present in all cortex extracts examined, including those from
sham-operated animals (data not shown). Examination of extracts
prepared from animals 6 hours after reperfusion suggested that both tPA
and uPA activity were elevated ipsilateral to the stroke in PBS-treated
animals but that only uPA appeared to be elevated in
neuroserpin-treated brains (Figure 4A). However, by 72 hours tPA
activity appeared to return to baseline, indicating that the increase
in tPA activity is transient and that neuroserpin can reduce the extent
of this increase. In contrast, uPA-catalyzed activity, which was
relatively low in the 6-hour extracts, increased dramatically in
ipsilateral extracts of animals killed 72 hours after reperfusion, and
this increase was apparent in both control and neuroserpin-treated
brains. However, like both tPA and uPA at 6 hours, the amount of uPA
activity at 72 hours was significantly lower in neuroserpin-treated
animals compared to controls (Figure 4A). Quantitative image analysis
of these data indicated that by 6 hours following reperfusion
ipsilateral to the stroke in PBS-treated animals there was an
approximately 50% increase in tPA activity and an approximately 125%
increase in uPA activity relative to baseline levels (Figure 4B).
However, in neuroserpin-treated animals the increase of both PAs
ipsilateral to the stroke was markedly reduced, showing only an
approximately 50% increase for uPA and no significant increase in tPA
compared to baseline levels (Figure 4B). These results indicate that
there is an early and transient increase in tPA activity ipsilateral to
the stroke and that neuroserpin is able to block this increase.
Similarly, there is an early increase in uPA activity ipsilateral to
the stroke, but in contrast to tPA this increase is not transient and
continues to rise at least up to 72 hours after reperfusion, and is not blocked by treatment with neuroserpin but is only reduced compared to
the PBS-treated animals.
View larger version (34K):
[in this window]
[in a new window]
| Fig 4.
SDS-PAGE zymography of brain extracts
. (A) SDS-PAGE zymography of brain extracts. Lane 1 is human tPA, lane
2 is a rat kidney extract as a marker for rat uPA, lanes 3 through 6 are extracts of brain 6 hours after reperfusion, and lanes 7 through 10 are extracts 72 hours after reperfusion. Lanes 3 and 7 are ipsilateral
to the infarct of PBS- treated animals, lanes 4 and 8 are contralateral
to the infarct. Lanes 5 and 9 are ipsilateral to the infarct in
neuroserpin-treated animals and lanes 6 and 10 are contralateral. (B)
Quantitative image analysis of PA activity from SDS-PAGE zymography of
brain extracts 6 hours following reperfusion. The results represent the
average fold increase in either tPA or uPA activity ipsilateral to the
stroke relative to normal baseline PA activities contralateral to the
stroke. PBS and Ns represent animals treated with either PBS or
neuroserpin, respectively, and n 3 for each condition tested.
P  .05 relative to the contralateral activity are shown,
and errors represent SEM.
|
|
To examine the distribution of proteinase activity within the brains of
the PBS- and neuroserpin-treated animals, in situ zymography of frozen
brain sections was performed. These data demonstrate that, like the
SDS-PAGE zymography, all of the proteolytic activity detected in both
control and neuroserpin-treated brains was plasminogen dependent,
because no proteinase activity was observed in the absence of
plasminogen (Figure 5A,D). At 6 hours after
reperfusion, proteinase activity in all sections was primarily associated with the meningeal tissues of both ipsilateral and contralateral sides. This activity was also completely blocked by the
addition of anti-tPA antibodies, indicating that the majority of PA
activity within the brain at this time is tPA (data not shown). In
contrast, by 72 hours after reperfusion, there was a large increase in
plasminogen-dependent proteolytic activity ipsilateral to the stroke in
control animals (Figure 5B), and unlike the sections at 6 hours or the
72 hours contralateral side, this activity was not restricted to the
meninges and was not completely blocked by the addition of anti-tPA
antibodies to the plasminogen overlay (arrows in Figure 5B,C). In
neuroserpin-treated animals this zone of proteinase activity was
significantly smaller than in the untreated animals (Figure 5E,F). This
suggests that by 72 hours much of the plasminogen-dependent activity
within the region of the stroke was not tPA. Consistent with this, the
addition of anti-uPA antibodies to the plasminogen overlay markedly
reduced proteolysis within the area of the stroke while having no
effect on the proteolytic activity in the meningeal tissues
contralateral to the stroke (data not shown). This implies that within
the area of the infarct at 72 hours following reperfusion there is a
significant increase in uPA activity. These results also suggest that
there is not a large up-regulation of either tPA or uPA immediately following stroke; however, by 72 hours after reperfusion, uPA-catalyzed proteolysis is significantly increased specifically within the region
of the infarct. These results are also consistent with the SDS-PAGE
zymography and suggest that the lesser increase in uPA activity
observed by SDS-PAGE zymography in neuroserpin-treated animals may
simply reflect the smaller size of the infarct in this group and not a
direct inhibition of the up-regulation of uPA-activity by neuroserpin.
View larger version (131K):
[in this window]
[in a new window]
| Fig 5.
In situ zymography and immunohistochemical staining of
brain sections
. In situ zymography in panels A-F; panels A and D are developed
without plasminogen, and all other panels are developed with
plasminogen. Panels C and F also contain anti-tPA antibodies. The white
arrows indicate the area of the infarct (magnification × 3). (G)
Immunohistochemical staining of tPA 6 hours after reperfusion. The
black arrows indicate tPA-positive blood vessels. (H)
Immunohistochemical staining of uPA in the area of penumbra 72 hours
after reperfusion (magnification in G and H × 400).
|
|
Immunohistochemical staining for tPA indicated that by 6 hours after
reperfusion, tPA antigen was detected only within the vascular
endothelial cells and not within neuronal cells (Figure 5G). Consistent
with the relatively low levels of uPA activity at 6 hours, no uPA
staining could be detected in these sections (data not shown). However,
by 72 hours after reperfusion, uPA immunoreactivity was readily
detected, but only in the area of ischemic penumbra (Figure 5H). This
is consistent with the in situ zymography analysis demonstrating uPA
activity predominantly within the cortex and only at 72 hours after
reperfusion. Finally, at 72 hours in neuroserpin-treated animals, there
was a marked reduction in the overall area where uPA antigen was
detected but not in the intensity of the staining, compared to
PBS-treated animals (data not shown). This further suggests that the
reduced uPA activity observed by zymography was likely due to the
reduced size of the infarct in neuroserpin-treated animals.
Basement membrane degradation after cerebral ischemia
Because excitotoxin-induced laminin degradation has been
suggested to be mediated by tPA and to precede apoptotic cell
death,18 we examined the effect of stroke on laminin
immunoreactivity. For this analysis we used a monoclonal antibody that
does not react strongly with rat laminin in fixed tissue unless the
tissue is first proteolyzed to expose cryptic laminin epitopes. This is
shown in Figure 6 panels A and B, where
panel A shows a section of unproteolyzed rat cortex reacted with the
antibody and panel B shows an adjacent section that was first treated
with proteinase in vitro before reaction with the antibody. These
results indicate that in the absence of proteolysis this antibody does
not react with vascular laminin. However, after proteolysis there is a
strong reaction that appears to be localized to the vessels. Thus, this antibody provides an excellent tool to probe for partial proteolysis of
the basement membrane within fixed brain tissue. Examination of laminin
staining in cortical tissue as early as 10 minutes after reperfusion
indicated that even at this early time there was apparently significant
proteolysis of the basement membrane in control animals (Figure 6C).
However, in neuroserpin-treated animals the extent of laminin
proteolysis was significantly reduced such that only slight staining of
the vascular laminin was apparent (Figure 6D). This latter result was
not due to the absence of laminin in this tissue because treatment of
the sections with proteinase in vitro yielded staining
indistinguishable from that shown in Figure 6B (data not shown).
Vascular laminin staining was also observed at 6 hours after
reperfusion in control animals and, similar to the results at 10 minutes, treatment with neuroserpin significantly reduced this staining
(Figure 6E,F). Furthermore, by 6 hours after reperfusion laminin
staining was also observed within neurons in the area of cerebral
ischemia and, as with vascular laminin staining, was reduced by
neuroserpin treatment (Figure 6E,F). The neuronal staining most likely
represents new synthesis of laminin because in control animals not
subjected to stroke no laminin staining was observed in neurons either
with or without proteinase treatment (Figure 6A,B). Laminin staining
remained strong at 24 and 48 hours, but started to decrease by 72 hours. Also, at each time point the neuroserpin-treated animals showed significantly less immunoreactivity than control animals (data not
shown). These data suggest that there is a very early proteolytic event
that appears to act on the vascular basement membrane and that
neuroserpin treatment is able to reduce this proteolysis.
View larger version (208K):
[in this window]
[in a new window]
| Fig 6.
Immunohistochemical staining of laminin
. Panels A and B show normal cortex in an animal without stroke. Panel
A was developed with anti-laminin but without pretreatment in vitro of
the section with proteinase. Panel B is an adjacent section with in
vitro proteinase treatment. Panels C to F are from stroked animals and
developed with antilaminin and no proteinase treatment. Panels C and D
are 10 minutes after reperfusion and panels E and F are 6 hours after
reperfusion. Panels C and E represent PBS-treated and panels D and F
neuroserpin-treated animals (magnification × 100).
|
|
Apoptosis
Because cerebral ischemia has been suggested to induce apoptosis in
the ischemic penumbra, a good therapeutic strategy aimed at reducing
cell death after stroke should target the recovery of cells in this
area. To see if neuroserpin reduced infarct volume by preventing
penumbral apoptosis, tissue from untreated and neuroserpin-treated animals was stained by the TUNEL method (Figure
7A-C). The extent of apoptosis in these
sections was then quantified as described above and these data are
shown in Figure 7, panel D. The number of cells within a defined area
of the penumbra with apoptotic bodies after 72 hours of cerebral
ischemia was 22 ± 5 in untreated animals and decreased to
8 ± 2 in neuroserpin-treated animals (Figure 7D). This indicates
that neuroserpin significantly inhibits penumbral apoptosis. To see if
neuroserpin also blocked cell death at earlier times, apoptosis was
also quantified at 6, 24, and 48 hours. These data indicate that at all
times examined apoptosis was reduced by at least 50% with neuroserpin
treatment (Figure 7E). Finally, to test if neuroserpin had a direct
effect on apoptosis, 2 independent assays were performed. In the first,
neuroserpin was tested for its ability to block T-cell
receptor-mediated apoptosis of a T-cell hybridoma in
vitro.42 In the second assay, neuroserpin was tested for
its ability to directly inhibit caspase activity in extracts of B
lymphoma cells treated with anti-Fas IgG to induce apoptosis and
caspase activation.43 In both assays neuroserpin had no
effect on either apoptosis or caspase activity (data not shown). Taken
together, these results indicate that neuroserpin is not a direct
inhibitor of apoptosis, and therefore, it is likely that neuroserpin
blocks events before induction of apoptosis.
View larger version (62K):
[in this window]
[in a new window]
| Fig 7.
Neuronal apoptosis within the ischemic penumbra
. TUNEL staining ipsilateral of the infarct in PBS-treated (panels A
and B) and neuroserpin-treated (panel C) animals. In panel A, NC
indicates the necrotic core and P indicates the area of the penumbra.
Panels B and C are high magnification images of the penumbra in control
(panel B) and neuroserpin-treated animals (panel C). Examples of cells
considered to be apoptotic for the purposes of quantification are
indicated with the open arrows; cells considered as necrotic are
indicated with the closed arrows. Magnification in panel A is
× 40 and in panels B and C × 400. (D) Quantitative
analysis of apoptosis in the area of penumbra 72 hours after
reperfusion. Quantitation was performed as described in "Materials
and methods" and only cells with apoptotic bodies present (panel B)
were counted. Control represents animals injected with PBS (n = 6).
Neuroserpin indicates animals injected with neuroserpin (n = 6).
P values < .05 are shown, and errors represent SEM. (E)
Quantitation of apoptosis was performed as in panel D at the times
indicated, ( ) PBS-treated animals, ( ) neuroserpin-treated
animals.
|
|
| |
Discussion |
Neuroserpin, a natural inhibitor of tPA, is found almost exclusively
within the CNS36 and shows an early and significant increase in its expression within the area of ischemic penumbra in
response to stroke (Figure 2). Cerebral ischemia induces neuronal depolarization44 and releases excitotoxins,29
which in turn triggers the release of tPA.45,46 Because tPA
may be associated with increased neuronal loss in response to both
ischemia and excitotoxins,5,8,15,33 the increased local
expression of neuroserpin following ischemia may represent an innate
protective response to elevated tPA levels and suggests that
neuroserpin may be a naturally occurring neuronal survival factor.
Consistent with this hypothesis, neuroserpin treatment resulted in a
significant decrease in stroke volume relative to control animals.
Furthermore, only functionally active neuroserpin was able to reduce
infarct size, suggesting that inhibition of proteinase activity was
necessary for the neuroprotective effects of neuroserpin (Figure 3).
Zymographic analysis of brain extracts at 6 and 72 hours after
reperfusion indicated that there was an early rise in both tPA and uPA
activity in the area of the infarct in control animals and that
treatment with neuroserpin significantly reduced these activities
(Figure 4). These data are similar to earlier results that reported an
increase in uPA activity in both rats and mice following cerebral
ischemia47,48 and to at least 1 other study that reported a
significant increase in tPA activity.5 However, in 2 studies tPA activity after stroke was reported to be either decreased47 or unchanged.48 The apparent
difference in the activity of tPA noted here compared to these earlier
studies may in part reflect the time after cerebral ischemia when tPA
was measured because our data suggest that the increase in tPA activity is transient and because none of these other studies measured tPA at 6 hours following reperfusion. These differences might also be due to the
different animal models used in the various studies. For example, the
model used here37,49 creates a permanent occlusion of the
middle cerebral artery at its crossing point with the inferior cerebral
vein with reperfusion provided by temporary clamping of the left
carotid artery. This produces an ischemic injury in a well-defined area
of the cerebral cortex (Figure 1), and in contrast to the intravascular
filament model,50 avoids the potential of large lesions to
the vascular endothelium and severe disruption of the blood-brain
barrier that could lead to significant changes in the local tPA
activity. Nonetheless, our results and those of Wang et al5
suggest that there is an early local increase in tPA activity in the
area of the infarct, and the data reported here further suggest that
this increase is transient. Because treatment with functionally active
neuroserpin reduced both the local increase in tPA activity as well as
the infarct size, it is possible that these 2 effects are related
and that by blocking the action of tPA very early after reperfusion the later increase of the infarcted area is prevented.
In contrast to tPA, uPA activity increased to very high levels by 72 hours following reperfusion and was localized almost exclusively to the
ischemic penumbra (Figures 4 and 5). The role of uPA after cerebral
ischemia is largely unknown. However, because the necrotic core is
already well defined by 72 hours after the stroke, it is unlikely that
the late increase in uPA activity plays an important role in the
development of the infarct. This inference is also consistent with a
recent study of stroke in uPA-deficient mice that indicated that there
was no difference in infarct volume between wild-type and
uPA/ mice 24 hours after reperfusion.8
However, because uPA has been demonstrated in both glial cells during
myelination and in mature cortical neurons,51 the late
expression of uPA activity and antigen suggests that uPA could
participate in the process of neuronal recovery after stroke as was
suggested by Rosenberg et al.47
Although the role of tPA activity in infarct evolution is not well
understood, tPA-induced plasmin cleavage of basement membrane laminin
has been suggested to play a role in excitotoxin-induced neuronal death
within the hippocampus18 and in the disappearance of
basement membrane antigens following ischemia and
reperfusion.52 The basement membrane is a specialized part
of the ECM that connects the endothelial cell compartment to the
surrounding cell layers.53 Laminins are important
components of the basement membrane, playing a pivotal role in cell-ECM
interactions, including promotion of neurite outgrowth, cell
attachment, proliferation, and differentiation, as well as in the
development and regeneration of the CNS.54-58 In the
present study exposure of cryptic laminin epitopes within the basement
membrane was observed within 10 minutes of reperfusion, suggesting that
there is proteolytic activity acting on the basement membrane very
early following cerebral ischemia (Figure 6). Like the observed
increase in tPA, this activity appears to be transient with peak
epitope exposure occurring within 6 to 24 hours of reperfusion. Whether
this effect is due to the direct action of tPA on laminin, is mediated
through plasmin as has been suggested,18 or involves matrix
metalloproteinases (MMPs)59 or other as yet unidentified proteinases is not clear. Regardless of which proteinase is responsible for the apparent basement membrane degradation, the extent of laminin
epitope expression was significantly decreased in neuroserpin-treated animals (Figure 6). This suggests that neuroserpin is inhibiting the
proteolytic attack on the basement membrane, most likely by inhibiting
tPA. Thus, neuroserpin, by blocking the early increase in tPA activity,
may be able to preserve the integrity of the basement membrane and thus
the blood-brain barrier after stroke.
It is known that disruption of cell-matrix interactions can lead to
apoptosis.60,61 Because the cryptic laminin epitopes were
observed as early as 10 minutes after reperfusion, with high levels of
neuronal expression seen by 6 hours (Figure 6), well before 24 to
48 hours, the peak of apoptosis (Figure 7E), the proteolytic
disruption of the basement membrane may be the trigger that
initiates the program of apoptotic neuronal cell death. Thus, the
capacity of active neuroserpin to block tPA-induced degradation of the
basement membrane may explain the ability of neuroserpin treatment to reduce neuronal apoptosis by nearly 70% (Figure 7D). Finally, although it has been demonstrated that apoptotic cell death in stroke is mediated by proteinases known as
caspases,62-64 neuroserpin failed to inhibit either caspase
activity or T-cell apoptosis, suggesting that neuroserpin is not an
inhibitor of apoptosis per se and does not directly block caspase
activation or activity.
Intraneuronal laminin-like immunoreactivity has been reported in both
the developing and adult CNS,33,65,66 and in astrocytes after transient ischemia.67 Laminin has also been shown to
promote neurite outgrowth.55 We observed morphologically
healthy neurons that were positive for intracellular laminin staining
in the area of penumbra in control animals by 6 hours after reperfusion
(Figure 6E), suggesting a role for intraneuronal laminin in neuronal
maintenance following ischemia. It is possible that synthesis of
laminin by neurons is in response to laminin degradation within the
basement membrane and that neuroserpin treated animals show fewer
laminin-positive cells because there is less basement membrane
degradation and thus many fewer distressed cells. It is interesting to
note that the region of the cortex that shows many laminin-positive
cells at 6 hours after reperfusion is the same region that shows many apoptotic cells at 48 hours. This suggests that if laminin expression is an early marker for cell distress then neuroserpin treatment must be
acting very early in the pathway that leads to neuronal apoptosis.
Taken together the data presented here suggest a model for
stroke-induced neuronal death within the ischemic penumbra and demonstrate the potential therapeutic benefits of neuroserpin in this
setting. We speculate that one of the first potentially deleterious
events to occur is the release of tPA by the vascular endothelial cells
in response to the acute ischemia. If there is also increased vascular
permeability at this time as a result of damage to the blood-brain
barrier,68 then this will allow tPA to cross from the lumen
of the vessel into the subvascular space, where it can bind directly to
laminin.69 This inappropriately targeted tPA can then,
either by itself or in concert with other proteinases such as plasmin
or MMPs,59,70 begin to degrade the basement membrane. This
leads to a further increase in vascular permeability, which in turn may
accelerate the degradation of the blood-brain barrier. In addition,
neuronal cells that are also dependent on the integrity of the basement
membrane may begin to lose their contacts with the substrate, which in
itself might induce a program of apoptosis as has been described for
other cell types.60 Our data also suggest that by
approximately 72 hours after the stroke onset the basement membrane
degradation and neuronal apoptosis have decreased, stabilizing the
lesion. At this time other factors such as uPA are up-regulated
possibly as part of the recovery process. Neuroserpin treatment, by
blocking the early effects of proteinase activity, may help to maintain the integrity of the basement membrane, preventing further passage of
tPA or other potentially harmful blood-borne factors to the subvascular
space. Neuroserpin may also directly prevent neuronal loss by helping
to preserve neuronal contacts to the basement membrane. Further studies
of the efficacy of neuroserpin in the treatment of acute stroke will
certainly clarify and refine this possible model.
| |
Acknowledgment |
We wish to thank K. Ingham for critically reading the manuscript.
| |
Footnotes |
Submitted November 18, 1999; accepted March 10, 2000.
Supported by National Institutes of Health Grants HL55374 and HL55747
to D.A.L.
Reprints: Daniel A. Lawrence, Biochemistry Department, J. H. Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way,
Rockville, MD 20855; e-mail: lawrenced{at}usa.redcross.org.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
The World Health Report 1999.
Making a Difference. 1-121. Geneva, Switzerland: The World Health Organization; 1999.
2.
Thorvaldsen P, Asplund K, Kuulasmaa K, Rajakangas AM, Schroll M.
Stroke incidence, case fatality, and mortality in the WHO MONICA project. World Health Organization Monitoring Trends and Determinants in Cardiovascular Disease.
Stroke.
1995;26:361-367[Abstract/Free Full Text].
3.
Caplan LR, Mohr JP, Kistler JP, Koroshetz W.
Should thrombolytic therapy be the first-line treatment for acute ischemic stroke? Thrombolysis not a panacea for ischemic stroke.
N Engl J Med.
1997;337:1309-1310[Free Full Text].
4.
The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group.
Tissue plasminogen activator for acute ischemic stroke.
N Engl J Med.
1995;333:1581-1587[Abstract/Free Full Text].
5.
Wang YF, Tsirka SE, Strickland S, Stieg PE, Soriano SG, Lipton SA.
Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice.
Nat Med.
1998;4:228-231[Medline]
[Order article via Infotrieve].
6.
Tabrizi P, Wang L, Seeds N, et al.
Tissue plasminogen activator (tPA) deficiency exacerbates cerebrovascular fibrin deposition and brain injury in a murine stroke model: studies in tPA-deficient mice and wild-type mice on a matched genetic background.
Arterioscler Thromb Vasc Biol.
1999;19:2801-2806[Abstract/Free Full Text].
7.
Klein GM, Li H, Sun P, Buchan AM.
Tissue plasminogen activator does not increase neuronal damage in rat models of global and focal ischemia.
Neurology.
1999;52:1381-1384[Abstract/Free Full Text].
8.
Nagai N, De Mol M, Lijnen HR, Carmeliet P, Collen D.
Role of plasminogen system components in focal cerebral ischemic infarction: a gene targeting and gene transfer study in mice.
Circulation.
1999;99:2440-2444[Abstract/Free Full Text].
9.
Qian Z, Gilbert ME, Colicos MA, Kandel ER, Kuhl D.
Tissue-plasminogen activator is induced as an immediate-early gene during seizure, kindling and long-term potentiation.
Nature.
1993;361:453-457[Medline]
[Order article via Infotrieve].
10.
Seeds NW, Williams BL, Bickford PC.
Tissue plasminogen activator induction in Purkinje neurons after cerebellar motor learning.
Science.
1995;270:1992-1994[Abstract/Free Full Text].
11.
Carroll PM, Tsirka SE, Richards WG, Frohman MA, Strickland S.
The mouse tissue plasminogen activator gene 5' flanking region directs appropriate expression in development and a seizure- enhanced response in the CNS.
Development
1994;120:3173-3183[Abstract].
12.
Friedman GC, Seeds NW.
Tissue plasminogen activator expression in the embryonic nervous system.
Brain Res Dev Brain Res.
1994;81:41-49[Medline]
[Order article via Infotrieve].
13.
Ware JH, Dibenedetto AJ, Pittman RN.
Localization of tissue plasminogen activator mRNA in adult rat brain.
Brain Res Bull.
1995;37:275-281[Medline]
[Order article via Infotrieve].
14.
Sappino AP, Madani R, Huarte J, et al.
Extracellular proteolysis in the adult murine brain.
J Clin Invest.
1993;92:679-685.
15.
Tsirka SE, Gualandris A, Amaral DG, Strickland S.
Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator.
Nature.
1995;377:340-344[Medline]
[Order article via Infotrieve].
16.
Tsirka SE, Rogove AD, Strickland S.
Neuronal cell death and tPA.
Nature.
1996;384:123-124[Medline]
[Order article via Infotrieve].
17.
Tsirka SE, Rogove AD, Bugge TH, Degen JL, Strickland S.
An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus.
J Neurosci.
1997;17:543-552[Abstract/Free Full Text].
18.
Chen ZL, Strickland S.
Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin.
Cell.
1997;91:917-925[Medline]
[Order article via Infotrieve].
19.
Symon L.
The relationship between CBF, evoked potentials and the clinical features in cerebral ischaemia.
Acta Neurol Scand Suppl.
1980;78:175-190[Medline]
[Order article via Infotrieve].
20.
Hakim AM.
The cerebral ischemic penumbra.
Can J Neurol Sci.
1987;14:557-559[Medline]
[Order article via Infotrieve].
21.
Hossmann KA.
Viability thresholds and the penumbra of focal ischemia.
Ann Neurol.
1994;36:557-565[Medline]
[Order article via Infotrieve].
22.
Pierce AR, Lo EH, Mandeville JB, Gonzalez RG, Rosen BR, Wolf GL.
MRI measurements of water diffusion and cerebral perfusion: their relationship in a rat model of focal cerebral ischemia.
J Cereb Blood Flow Metab.
1997;17:183-190[Medline]
[Order article via Infotrieve].
23.
Grohn OH, Lukkarinen JA, Oja JM, et al.
Noninvasive detection of cerebral hypoperfusion and reversible ischemia from reductions in the magnetic resonance imaging relaxation time, T2.
J Cereb Blood Flow Metab.
1998;18:911-920[Medline]
[Order article via Infotrieve].
24.
Hoehn-Berlage M, Norris DG, Kohno K, Mies G, Leibfritz D, Hossmann KA.
Evolution of regional changes in apparent diffusion coefficient during focal ischemia of rat brain: the relationship of quantitative diffusion NMR imaging to reduction in cerebral blood flow and metabolic disturbances.
J Cereb Blood Flow Metab.
1995;15:1002-1011[Medline]
[Order article via Infotrieve].
25.
Benveniste H, Drejer J, Schousboe A, Diemer NH.
Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis.
J Neurochem.
1984;43:1369-1374[Medline]
[Order article via Infotrieve].
26.
Globus MY, Busto R, Dietrich WD, Martinez E, Valdes I, Ginsberg MD.
Effect of ischemia on the in vivo release of striatal dopamine, glutamate, and gamma-aminobutyric acid studied by intracerebral microdialysis.
J Neurochem.
1988;51:1455-1464[Medline]
[Order article via Infotrieve].
27.
Miyashita K, Abe H, Nakajima T, et al.
Glutamate release in the gerbil hippocampus after middle cerebral artery occlusion.
Neuroreport.
1994;5:945-948[Medline]
[Order article via Infotrieve].
28.
Uchiyama-Tsuyuki Y, Araki H, Yae T, Otomo S.
Changes in the extracellular concentrations of amino acids in the rat striatum during transient focal cerebral ischemia.
J Neurochem.
1994;62:1074-1078[Medline]
[Order article via Infotrieve].
29.
Takagi K, Ginsberg MD, Globus MY, et al.
Changes in amino acid neurotransmitters and cerebral blood flow in the ischemic penumbral region following middle cerebral artery occlusion in the rat: correlation with histopathology.
J Cereb Blood Flow Metab.
1993;13:575-585[Medline]
[Order article via Infotrieve].
30.
Li Y, Chopp M, Jiang N, Yao F, Zaloga C.
Temporal profile of in situ DNA fragmentation after transient middle cerebral artery occlusion in the rat.
J Cereb Blood Flow Metab.
1995;15:389-397[Medline]
[Order article via Infotrieve].
31.
Linnik MD, Zobrist RH, Hatfield MD.
Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats.
Stroke.
1993;24:2002-2008[Abstract/Free Full Text].
32.
Linnik MD, Miller JA, Sprinkle-Cavallo J, et al.
Apoptotic DNA fragmentation in the rat cerebral cortex induced by permanent middle cerebral artery occlusion.
Brain Res Mol Brain Res.
1995;32:116-124[Medline]
[Order article via Infotrieve].
33.
Nagai N, Urano T, Endo A, Takahashi H, Takada Y, Takada A.
Neuronal degeneration and a decrease in laminin-like immunoreactivity is associated with elevated tissue-type plasminogen activator in the rat hippocampus after kainic acid injection.
Neurosci Res.
1999;33:147-154[Medline]
[Order article via Infotrieve].
34.
Osterwalder T, Contartese J, Stoeckli ET, Kuhn TB, Sonderegger P.
Neuroserpin, an axonally secreted serine protease inhibitor.
EMBO J.
1996;15:2944-2953[Medline]
[Order article via Infotrieve].
35.
Schrimpf SP, Bleiker AJ, Brecevic L, et al.
Human neuroserpin (PI12): cDNA cloning and chromosomal localization to 3q26.
Genomics.
1997;40:55-62[Medline]
[Order article via Infotrieve].
36.
Hastings GA, Coleman TA, Haudenschild CC, et al.
Neuroserpin, a brain-associated inhibitor of tissue plasminogen activator is localized primarily in neurons. Implications for the regulation of motor learning and neuronal survival.
J Biol Chem.
1997;272:33062-33067[Abstract/Free Full Text].
37.
Tamura A, Graham DI, McCulloch J, Teasdale GM.
Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion.
J Cereb Blood Flow Metab.
1981;1:53-60[Medline]
[Order article via Infotrieve].
38.
Brint S, Jacewicz M, Kiessling M, Tanabe J, Pulsinelli W.
Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries.
J Cereb Blood Flow Metab.
1988;8:474-485[Medline]
[Order article via Infotrieve].
39.
Osborne KA, Shigeno T, Balarsky AM, et al.
Quantitative assessment of early brain damage in a rat model of focal cerebral ischaemia.
J Neurol Neurosurg Psychiatry.
1987;50:402-410[Abstract/Free Full Text].
40.
MacManus JP, Buchan AM, Hill IE, Rasquinha I, Preston E.
Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain.
Neurosci. Lett.
1993;164:89-92[Medline]
[Order article via Infotrieve].
41.
Li Y, Sharov VG, Jiang N, Zaloga C, Sabbah HN, Chopp M.
Ultrastructural and light microscopic evidence of apoptosis after middle cerebral artery occlusion in the rat.
Am J Pathol.
1995;146:1045-1051[Abstract].
42.
Shi YF, Szalay MG, Paskar L, et al.
Activation-induced cell death in T cell hybridomas is due to apoptosis. Morphologic aspects and DNA fragmentation.
J Immunol.
1990;144:3326-3333[Abstract].
43.
Stennicke HR, Salvesen GS.
Biochemical characteristics of caspases-3, -6, -7, and -8.
J Biol Chem.
1997;272:25719-25723[Abstract/Free Full Text].
44.
Pulsinelli A, Jacewicz M.
Animal models of brain ischemia. In:
Barnett HJM,Mohr JP,Bennett S,Yatsu F, eds.
Stroke. Pathophysiology, Diagnosis and Management. New York: Churchill Livingstone; 1992:49-68.
45.
Gualandris A, Jones TE, Strickland S, Tsirka SE.
Membrane depolarization induces calcium-dependent secretion of tissue plasminogen activator.
J Neurosci.
1996;16:2220-2225[Abstract/Free Full Text].
46.
Parmer RJ, Mahata M, Mahata S, Sebald MT, O'Connor DT, Miles LA.
Tissue plasminogen activator (t-PA) is targeted to the regulated secretory pathway: catecholamine storage vesicles as a reservoir for the rapid release of t-PA.
J Biol Chem.
1997;272:1976-1982[Abstract/Free Full Text].
47.
Rosenberg GA, Navratil M, Barone F, Feuerstein G.
Proteolytic cascade enzymes increase in focal cerebral ischemia in rat.
J Cereb Blood Flow Metab.
1996;16:360-366[Medline]
[Order article via Infotrieve].
48.
Ahn MY, Zhang ZG, Tsang W, Chopp M.
Endogenous plasminogen activator expression after embolic focal cerebral ischemia in mice.
Brain Res.
1999;837:169-176[Medline]
[Order article via Infotrieve].
49.
Tamura A, Graham DI, McCulloch J, Teasdale GM.
Focal cerebral ischaemia in the rat: 2. Regional cerebral blood flow determined by [14C]iodoantipyrine autoradiography following middle cerebral artery occlusion.
J Cereb Blood Flow Metab.
1981;1:61-69[Medline]
[Order article via Infotrieve].
50.
Longa EZ, Weinstein PR, Carlson S, Cummins R.
Reversible middle cerebral artery occlusion without craniectomy in rats.
Stroke.
1989;20:84-91[Abstract/Free Full Text].
51.
Del Bigio MR, Jacque CM.
Localization of proteinase expression in the developing rabbit brain.
Brain Res Dev Brain Res.
1995;86:345-347[Medline]
[Order article via Infotrieve].
52.
Hamann GF, Okada Y, Fitridge R, del Zoppo GJ.
Microvascular basal lamina antigens disappear during cerebral ischemia and reperfusion.
Stroke.
1995;26:2120-2126[Abstract/Free Full Text].
53.
Yurchenco PD, Schittny JC.
Molecular architecture of basement membranes.
FASEB J.
1990;4:1577-1590[Abstract].
54.
Paulsson M.
Basement membrane proteins: structure, assembly, and cellular interactions.
Crit Rev Biochem Mol Biol.
1992;27:93-127[Medline]
[Order article via Infotrieve].
55.
Calof AL, Campanero MR, O'Rear JJ, Yurchenco PD, Lander AD.
Domain-specific activation of neuronal migration and neurite outgrowth- promoting activities of laminin.
Neuron.
1994;13:117-130[Medline]
[Order article via Infotrieve].
56.
Hammarback JA, McCarthy JB, Palm SL, Furcht LT, Letourneau PC.
Growth cone guidance by substrate-bound laminin pathways is correlated with neuron-to-pathway adhesivity.
Dev Biol.
1988;126:29-39[Medline]
[Order article via Infotrieve].
57.
Liesi P.
Laminin-immunoreactive glia distinguish regenerative adult CNS systems from non-regenerative ones.
EMBO J.
1985;4:2505-2511[Medline]
[Order article via Infotrieve].
58.
Millaruelo AI, Nieto-Sampedro M, Cotman CW.
Cooperation between nerve growth factor and laminin or fibronectin in promoting sensory neuron survival and neurite outgrowth.
Brain Res.
1988;466:219-228[Medline]
[Order article via Infotrieve].
59.
Rosenberg GA, Estrada EY, Dencoff JE.
Matrix metalloproteinases and TIMPs are associated with blood-brain barrier opening after reperfusion in rat brain.
Stroke.
1998;29:2189-2195[Abstract/Free Full Text].
60.
Meredith JEJ, Fazeli B, Schwartz MA.
The extracellular matrix as a cell survival factor.
Mol Biol Cell.
1993;4:953-961[Abstract].
61.
Murtomaki S, Trenkner E, Wright JM, Saksela O, Liesi P.
Increased proteolytic activity of the granule neurons may contribute to neuronal death in the weaver mouse cerebellum.
Dev Biol.
1995;168:635-648[Medline]
[Order article via Infotrieve].
62.
Barinaga M.
Death by dozens of cuts.
Science.
1998;280:32-34[Free Full Text].
63.
Cheng Y, Deshmukh M, D'Costa A, et al.
Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury.
J Clin Invest.
1998;101:1992-1999[Medline]
[Order article via Infotrieve].
64.
Namura S, Zhu J, Fink K, et al.
Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia.
J Neurosci.
1998;18:3659-3668[Abstract/Free Full Text].
65.
Hagg T, Portera-Cailliau C, Jucker M, Engvall E.
Laminins of the adult mammalian CNS; laminin-alpha2 (merosin M-) chain immunoreactivity is associated with neuronal processes.
Brain Res.
1997;764:17-27[Medline]
[Order article via Infotrieve].
66.
Suzuki H, Yamamoto T, Yamamoto H, et al.
Intraneuronal laminin-like immunoreactivity in the human central nervous system.
Brain Res.
1990;520:324-329[Medline]
[Order article via Infotrieve].
67.
Jucker M, Bialobok P, Kleinman HK, Walker LC, Hagg T, Ingram DK.
Laminin-like and laminin-binding protein-like immunoreactive astrocytes in rat hippocampus after transient ischemia. Antibody to laminin-binding protein is a sensitive marker of neural injury and degeneration.
Ann N Y Acad Sci.
1993;679:245-252[Medline]
[Order article via Infotrieve].
68.
Kogure K, Kato H.
Neurochemistry of stroke. In:
Barnett HJM,Mohr JP,Bennett S,Yatsu F, eds.
Stroke. Pathophysiology, Diagnosis and Management. New York: Churchill Livingstone; 1992:69-102.
69.
Salonen EM, Zitting A, Vaheri A.
Laminin interacts with plasminogen and its tissue-type activator.
FEBS Lett.
1984;172:29-32[Medline]
[Order article via Infotrieve].
70.
Heo JH, Lucero J, Abumiya T, Koziol JA, Copeland BR, del Zoppo GJ.
Matrix metalloproteinases increase very early during experimental focal cerebral ischemia.
J Cereb Blood Flow Metab.
1999;19:624-633[Medline]
[Order article via Infotrieve].
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
A. L. Samson, R. J. Borg, B. Niego, C. H. Y. Wong, P. J. Crack, T. Yongqing, and R. L. Medcalf
A nonfibrin macromolecular cofactor for tPA-mediated plasmin generation following cellular injury
Blood,
August 27, 2009;
114(9):
1937 - 1946.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Yang, N. Nemkul, A. Shereen, A. Jone, R. S. Dunn, D. A. Lawrence, D. Lindquist, and C.-Y. Kuan
Therapeutic Administration of Plasminogen Activator Inhibitor-1 Prevents Hypoxic-Ischemic Brain Injury in Newborns
J. Neurosci.,
July 8, 2009;
29(27):
8669 - 8674.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Iwata, T. Okamura, N. Ishibashi, D. Zurakowski, H. G.W. Lidov, and R. A. Jonas
Optimal dose of aprotinin for neuroprotection and renal function in a piglet survival model.
J. Thorac. Cardiovasc. Surg.,
June 1, 2009;
137(6):
1521 - 1529.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Zhang, J. An, D. K. Strickland, and M. Yepes
The Low-Density Lipoprotein Receptor-Related Protein 1 Mediates Tissue-Type Plasminogen Activator-Induced Microglial Activation in the Ischemic Brain
Am. J. Pathol.,
February 1, 2009;
174(2):
586 - 594.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Takasawa, I. Kato, K. Takasawa, Y. Ishii, T. Yoshida, M. H. Shehata, H. Kawaguchi, O. M. M. Mohafez, M. Sasahara, and K. Hiraga
Mutation-, Aging-, and Gene Dosage-dependent Accumulation of Neuroserpin (G392E) in Endoplasmic Reticula and Lysosomes of Neurons in Transgenic Mice
J. Biol. Chem.,
December 19, 2008;
283(51):
35606 - 35613.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Lebeurrier, S. Launay, R. Macrez, E. Maubert, H. Legros, A. Leclerc, S. P. Jamin, J.-Y. Picard, S. Marret, V. Laudenbach, et al.
Anti-Mullerian-hormone-dependent regulation of the brain serine-protease inhibitor neuroserpin
J. Cell Sci.,
October 15, 2008;
121(20):
3357 - 3365.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. An, C. Zhang, R. Polavarapu, X. Zhang, X. Zhang, and M. Yepes
Tissue-type plasminogen activator and the low-density lipoprotein receptor-related protein induce Akt phosphorylation in the ischemic brain
Blood,
October 1, 2008;
112(7):
2787 - 2794.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Danielyan, K. Ganguly, B.-S. Ding, D. Atochin, S. Zaitsev, J.-C. Murciano, P. L. Huang, S. E. Kasner, D. B. Cines, and V. R. Muzykantov
Cerebrovascular Thromboprophylaxis in Mice by Erythrocyte-Coupled Tissue-Type Plasminogen Activator
Circulation,
September 30, 2008;
118(14):
1442 - 1449.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Polavarapu, J. An, C. Zhang, and M. Yepes
Regulated Intramembrane Proteolysis of the Low-Density Lipoprotein Receptor-Related Protein Mediates Ischemic Cell Death
Am. J. Pathol.,
May 1, 2008;
172(5):
1355 - 1362.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Zhang, R. Polavarapu, H. She, Z. Mao, and M. Yepes
Tissue-Type Plasminogen Activator and the Low-Density Lipoprotein Receptor-Related Protein Mediate Cerebral Ischemia-Induced Nuclear Factor-{kappa}B Pathway Activation
Am. J. Pathol.,
October 1, 2007;
171(4):
1281 - 1290.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Lehmensiek, S. Sussmuth, G. Tauscher, J. Brettschneider, S. Felk, F. Gillardon, and H. Tumani
Cerebrospinal fluid proteome profile in multiple sclerosis
Multiple Sclerosis,
August 1, 2007;
13(7):
840 - 849.
[Abstract]
[PDF]
|
|
|
|
|
|
|
R. Polavarapu, M. C. Gongora, H. Yi, S. Ranganthan, D. A. Lawrence, D. Strickland, and M. Yepes
Tissue-type plasminogen activator-mediated shedding of astrocytic low-density lipoprotein receptor-related protein increases the permeability of the neurovascular unit
Blood,
April 15, 2007;
109(8):
3270 - 3278.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. W. Weiss, A. L. Samson, B. Niego, P. B. Daniel, and R. L. Medcalf
Oncostatin M is a neuroprotective cytokine that inhibits excitotoxic injury in vitro and in vivo
FASEB J,
November 1, 2006;
20(13):
2369 - 2371.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Simonin, Y. Charron, P. Sonderegger, J.-D. Vassalli, and A. C. Kato
An Inhibitor of Serine Proteases, Neuroserpin, Acts as a Neuroprotective Agent in a Mouse Model of Neurodegenerative Disease
J. Neurosci.,
October 11, 2006;
26(41):
10614 - 10619.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. J. Kinghorn, D. C. Crowther, L. K. Sharp, C. Nerelius, R. L. Davis, H. T. Chang, C. Green, D. C. Gubb, J. Johansson, and D. A. Lomas
Neuroserpin Binds Abeta and Is a Neuroprotective Component of Amyloid Plaques in Alzheimer Disease
J. Biol. Chem.,
September 29, 2006;
281(39):
29268 - 29277.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. M. de Groot and G. J. M. Martens
Expression of Neuroserpin Is Linked to Neuroendocrine Cell Activation
Endocrinology,
September 1, 2005;
146(9):
3791 - 3799.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Onda, D. Belorgey, L. K. Sharp, and D. A. Lomas
Latent S49P Neuroserpin Forms Polymers in the Dementia Familial Encephalopathy with Neuroserpin Inclusion Bodies
J. Biol. Chem.,
April 8, 2005;
280(14):
13735 - 13741.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Yepes, S. A.N. Brown, E. G. Moore, E. P. Smith, D. A. Lawrence, and J. A. Winkles
A Soluble Fn14-Fc Decoy Receptor Reduces Infarct Volume in a Murine Model of Cerebral Ischemia
Am. J. Pathol.,
February 1, 2005;
166(2):
511 - 520.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Yepes and D. A. Lawrence
New Functions for an Old Enzyme: Nonhemostatic Roles for Tissue-Type Plasminogen Activator in the Central Nervous System
Experimental Biology and Medicine,
December 1, 2004;
229(11):
1097 - 1104.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Miranda, K. Romisch, and D. A. Lomas
Mutants of Neuroserpin That Cause Dementia Accumulate as Polymers within the Endoplasmic Reticulum
J. Biol. Chem.,
July 2, 2004;
279(27):
28283 - 28291.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B.-Q. Zhao, Y. Ikeda, H. Ihara, T. Urano, W. Fan, S. Mikawa, Y. Suzuki, K. Kondo, K. Sato, N. Nagai, et al.
Essential role of endogenous tissue plasminogen activator through matrix metalloproteinase 9 induction and expression on heparin-produced cerebral hemorrhage after cerebral ischemia in mice
Blood,
April 1, 2004;
103(7):
2610 - 2616.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Makarova, I. Mikhailenko, T. H. Bugge, K. List, D. A. Lawrence, and D. K. Strickland
The Low Density Lipoprotein Receptor-related Protein Modulates Protease Activity in the Brain by Mediating the Cellular Internalization of Both Neuroserpin and Neuroserpin-Tissue-type Plasminogen Activator Complexes
J. Biol. Chem.,
December 12, 2003;
278(50):
50250 - 50258.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Gveric, B. Herrera, A. Petzold, D. A. Lawrence, and M. L. Cuzner
Impaired fibrinolysis in multiple sclerosis: a role for tissue plasminogen activator inhibitors
Brain,
July 1, 2003;
126(7):
1590 - 1598.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Lotem, H. Gal, R. Kama, N. Amariglio, G. Rechavi, E. Domany, L. Sachs, and D. Givol
Inhibition of p53-induced apoptosis without affecting expression of p53-regulated genes
PNAS,
May 27, 2003;
100(11):
6718 - 6723.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Barker-Carlson, D. A. Lawrence, and B. S. Schwartz
Acyl-Enzyme Complexes between Tissue-type Plasminogen Activator and Neuroserpin are Short-lived in Vitro
J. Biol. Chem.,
November 27, 2002;
277(49):
46852 - 46857.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Zhang, L. Zhang, M. Yepes, Q. Jiang, Q. Li, P. Arniego, T. A. Coleman, D. A. Lawrence, and M. Chopp
Adjuvant Treatment With Neuroserpin Increases the Therapeutic Window for Tissue-Type Plasminogen Activator Administration in a Rat Model of Embolic Stroke
Circulation,
August 6, 2002;
106(6):
740 - 745.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Cuadrado, C. Navarro-Yubero, H. Furneaux, J. Kinter, P. Sonderegger, and A. Munoz
HuD binds to three AU-rich sequences in the 3'-UTR of neuroserpin mRNA and promotes the accumulation of neuroserpin mRNA and protein
Nucleic Acids Res.,
May 15, 2002;
30(10):
2202 - 2211.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Belorgey, D. C. Crowther, R. Mahadeva, and D. A. Lomas
Mutant Neuroserpin (S49P) That Causes Familial Encephalopathy with Neuroserpin Inclusion Bodies Is a Poor Proteinase Inhibitor and Readily Forms Polymers in Vitro
J. Biol. Chem.,
May 3, 2002;
277(19):
17367 - 17373.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. A. McMahon, E. Petitclerc, S. Stefansson, E. Smith, M. K. K. Wong, R. J. Westrick, D. Ginsburg, P. C. Brooks, and D. A. Lawrence
Plasminogen Activator Inhibitor-1 Regulates Tumor Growth and Angiogenesis
J. Biol. Chem.,
August 31, 2001;
276(36):
33964 - 33968.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. A. Silverman, P. I. Bird, R. W. Carrell, F. C. Church, P. B. Coughlin, P. G. W. Gettins, J. A Irving, D. A. Lomas, C. J. Luke, R. W. Moyer, et al.
The Serpins Are an Expanding Superfamily of Structurally Similar but Functionally Diverse Proteins. EVOLUTION, MECHANISM OF INHIBITION, NOVEL FUNCTIONS, AND A REVISED NOMENCLATURE
J. Biol. Chem.,
August 31, 2001;
276(36):
33293 - 33296.
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction