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Université de Caen, CNRS UMR 6551, IFR 47, bd H. Becquerel, BP 5229, 14074 Caen Cedex, France; and
* Max-Planck-Institut, für physiologische und klinische Forschung, D-61231 Bad-Nauheim, Germany
1Correspondence: Université de CAEN-CNRS UMR 6551, Laboratoire de Neurosciences, BP 5229, 14074 Caen Cedex, France. E-mail: d.vivien{at}neuro.unicaen.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: TGF-ß • plasminogen activator • neuroserpin • NTN
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, which plays a
critical role in the metabolism of the extracellular matrix. t-PA
expression has been correlated with hippocampal function, and its mRNA
level is rapidly increased in relation with neuronal activity
(2)
. Furthermore, t-PA-deficient mice are resistant to
neuronal cell death induced either by excitotoxins (3)
or
ischemic injury (4)
. These data suggest that excessive
neuronal activity can lead to neuronal death through the t-PA
proteolytic loop.
Transforming growth factor-ß1 (TGF-ß1) is the prototype of a
large family of peptides, including BMPs (bone morphogenetic proteins)
and activin (5)
. TGF-ßs, BMPs, and activin elicit their
effects by binding to their own set of cell surface serine/threonine
kinase receptors (6
, 7)
expressed in cortical neurons and
astrocytes (8)
. The activated TGF-ß receptors (TßR-I
and TßR-II) induce the phosphorylation of two intracellular proteins,
Smad2 and Smad3 (9
, 10)
, which form hetero-oligomeric
complexes with Smad4 (11
, 12)
. These complexes translocate
to the nucleus, where they regulate transcriptional responses
(13)
. More recently, two novel members of the TGF-ß
family were identified in the CNS as GDNF (glial cell line-derived
neurotrophic factor) and neurturin (NTN), which transduce their signal
through a tyrosine kinase receptor, c-Ret (14
, 15)
.
TGF-ßs have been found to be abundantly expressed in the central
nervous system (CNS) and are characterized as injury-related peptide
growth factors (16
17
18
19
20
, 8)
. In addition to its potentially
neuroprotective effects (21
, 22)
, TGF-ß1 regulates
biological activity in astrocytes and microglia (18)
.
TGF-ß1 is known to activate the transcription of mammalian genes
important for cell cycle regulation, (23
24
25)
as well as
some involved in the extracellular matrix metabolism, such as the
plasminogen activator inhibitor type I (PAI-1), which controls the
activity of t-PA (26
27
28
29)
. Recently, a DNA element (CAGA
box) in the PAI-1 promoter was identified as that with which the
transcriptional factors Smad3 and Smad4 directly interact after TGF-ß
treatment (30)
. We demonstrated in vitro that
an up-regulation of PAI-1 in astrocytes mediates the neuroprotective
activity of TGF-ß1 against NMDA-induced excitotoxicity
(31)
.
These data indicate that 1) t-PA may play a critical role in neuronal death induced by excitotoxins; 2) TGF-ß1 regulates the neuronal cell death induced by excitotoxins; and 3) PAI-1 is a target for the action of TGF-ß. Based on this literature, as a first step we investigated the changes in serpins and t-PA expression induced by focal cerebral ischemia in mice. Second, we studied the effect of several members of the TGF-ß family such as TGF-ß, activins, BMPs, GDNF, and NTN on the regulation of these serpins in cultured cortical astrocytes and neurons.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell cultures
Cortical astrocyte cell cultures were prepared from 1- to 3-day
postnatal mice (32)
. Cerebral cortices were dissected and
incubated for 20–30 min in 0.025% trypsin in stock media (MS,
Eagle's MEM augmented with 2 mM glutamine and 25 mM glucose) and
transferred to MS supplemented with 10% fetal bovine and 10% horse
sera for trituration. Dissociated cells were plated at a density of
~3 x 105 cells per well. Cultures were
kept at 37°C in a humidified 5% CO2-containing
atmosphere. The medium was changed once weekly. Experiments were
performed on cultured cortical astrocytes after 21 days in
vitro.
Near-pure neuronal cell cultures, containing less than 5%
astrocytes, were prepared as previously detailed (33)
from
14- or 15-day-old embryonic mice. Dissociated cortical cells in MS
supplemented with 5% fetal bovine and 5% horse sera were plated in
multiwell vessels that had previously been coated with poly-D-lysine.
After 3 to 5 days in vitro, cultures were treated with 3
µM cytosine arabinoside in order to inhibit the proliferation of
astrocytes. There was no further exchange of the medium. Experiments
were performed on neuronal cultures after 12–13 days in
vitro.
Semiquantitative reverse transcriptase PCR
Total RNAs were prepared from cerebral cortex, cultured neurons,
and astrocytes by a phenol/chloroform extraction method using the RNA
Ble extraction kit (Eurobio, Paris, France). Samples (1 µg) of total
mRNA were transcribed into cDNA using poly-dT oligonucleotides. An
aliquot of the produced cDNAs was amplified using sense and antisense
primers for PAI-1, t-PA, NSP, PN-1, and ß-actin, respectively (see
Table 1
). For the semiquantitative experiments, an aliquot of cDNA libraries (1
µl from 2O µl) was amplified by PCR with specific oligonucleotides
for ß-actin (30 cycles of PCR were chosen corresponding to the 50%
of the saturation curve of the PCR products). PCR products were cloned
in pGEMT-4Z and their identity was confirmed by sequencing. Amplified
products were separated by agarose gel electrophoresis and visualized
by ethidium bromide staining. PCR experiments for t-PA, PAI-1, NSP, and
PN-1 were performed only if the expression pattern obtained for the
housekeeping gene ß-actin showed no significant modification between
our different samples.
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In situ hybridization
The technique used for in situ hybridization was
previously described by Breier et al. (34)
. Briefly, RNA
probes were generated by in vitro transcription of the
plasmid pGEMT4Z containing a partial mouse PAI-1 cDNA, spanning the
base pairs from 466 to 772. This partial cDNA was derived from our own
reverse transcriptase (RT) -PCR product, cloned in PGEMT4Z, and
characterized by sequencing. Single-stranded antisense or sense RNA
probes were generated using 100 µCi 35S-UTP and
T7 or SP6 RNA polymerases, respectively, as described by the
manufacturer (Stratagene, San Diego, Calif.). Frozen brains embedded in
tissue tec were sectioned in cryostat. Sections (10 µM) were melted
on silane-coated glass slides. Sections were incubated in 2x SSC at
70°C, digested with Pronase (40 µg/ml), fixed in 4%
paraformaldehyde, and acetylated with acetic anhydride diluted 1:400
with 0.1 M triethylamine. Hybridization was performed in buffer
containing 50% formamide, 10% dextran sulfate, 10 mM Tris-HCl (pH
7.5), 10 mM sodium phosphate (pH 6.8), 2x SSC, 5 mM EDTA, 150 µg/ml
yeast tRNA, 0.1 mM UTP, 1 mM ADPßS, 1 mM ATP
S, 10 mM
dithiothreitol, 10 mM 2-mercaptoethanol, and 2.5 x
104 cpm/ml 35S-labeled RNA
probe overnight at 48°C. Sections were washed in 2x SSC/50%
formamide at 37°C overnight and dehydrated in graded ethanol. Then
slices were coated with Kodak NTB-2 emulsion (Eastman Kodak, Rochester,
N.Y.) diluted 1:1 in water and exposed for 22 days. Slides were
developed and counterstained with Toluidine blue, air dried, and
mounted.
Western blot experiments
Cells were incubated for 6 h at 37°C in the presence of
TGF-ß1 (1 ng/ml). Cells were removed by washing three times in a
solution containing 10 mM Tris-HCl (pH 8.0), 0.5% sodium deoxycholate
with 1 mM phenylmethylsulfonyl fluoride. Cell matrix proteins were
extracted and sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was performed in the presence of dithiothreitol (DTT) and
ß-mercaptoethanol; thereafter, the proteins were transferred to a
polyvinylidene difluoride Imobilon-P transfer membrane filter
(Millipore, Bedford, Mass.). Membranes were blocked and probed with an
antibody raised against PAI-1 (sheep anti-mouse PAI-1; Orthoclinical,
France) and revealed by a combination of both biotinylated rabbit
anti-sheep antibody and a peroxidase-conjugated streptavidin reagent.
Blots were finally developed with an enhanced chemiluminescence Western
blotting detection system (NEN).
PAI-1 metabolic labeling
Cells were incubated in methionine/cysteine-free MEM containing
50 mCi/ml of
[35S]methionine/35S-cysteine
(Trans-label, NEN, 1100 Ci/mmol) for 6 h at 37°C in the presence
of TGF-ß1 (1 ng/ml). Cells were removed by washing three times in a
solution containing 10 mM Tris-HCl (pH 8.0), 0.5% sodium deoxycholate
with 1 mM phenylmethylsulfonyl fluoride. Cell matrix proteins were
extracted and SDS-PAGE electrophoresis was performed in the presence of
DTT and ß-mercaptoethanol.
Immunoprecipitation
Metabolically labeled cells were solubilized in 1 ml of lytic
buffer in the presence of protease inhibitors at 4°C for 30 min.
Through the use of specific antibodies, immunoprecipitations were
performed from the supernatant of the lysis solution for 2 h at
4°C and followed by adsorption to protein-G-Sepharose (Pharmacia
Biotech). Bound proteins were eluted in DTT and ß-mercaptoethanol
containing a loading sample buffer; SDS-PAGE electrophoresis was then
performed.
Transfection and luciferase assays
Cells were transiently transfected with the indicated constructs
using the lipofection method (Lipofectamine, Life Technologies) and
stimulated with 1 ng/ml of human recombinant TGF-ß1 (R&D, Abingdon,
U.K.). Luciferase activities were quantified 24 h later with a
commercial kit (Promega). Values were normalized with respect to the
Renilla luciferase activity as expressed by the cotransfected control
vector pRL-TK (Promega).
Immunocytochemistry
Cell cultures were fixed in the presence of 4%
paraformaldehyde/phosphate-buffered saline (PBS). The preparations were
then washed in PBS, followed by 20 min of treatment with PBS containing
1% of bovine serum albumin and 0.1% of Tween-20. Primary antibodies
were incubated overnight at 4°C, and the appropriate biotinylated
secondary antibodies were added for an additional 2 h. The cell
culture was incubated in the VECTASTIN ABC reagent and developed in a
substrate for peroxidase. Controls were performed by omitting primary
antibodies.
Surgical procedure
Surgical procedures were conducted according to national
legislation and as described previously by Gotti et al.
(35)
. Briefly, male wild-type mice (OF-1) weighing 20 to
25 g were anesthetized with an intraperitoneal chloral hydrate
injection (500 mg/kg, i.p.); then a skin incision was made between the
eye and the ear. The parotid gland and surrounding soft tissues were
reflected downward and the underlying temporalis was incised. The
tissue was retracted until the MCA (left middle cerebral artery) was
visible through the surface of the skull. A craniectomy was performed
and the MCA was coagulated. The temporalis and the parotid gland were
replaced and the incision sutured. Mice were placed in a warm
environment until they recovered from anesthesia. Extraction of total
RNA was performed as described above in both ipsilateral and
contralateral cerebral cortices and compared with sham-operated mice.
The infarcted volume estimated 24 h after the beginning of the
occlusion represents a reproducible lesion of 30
mm3 restricted to the cortex, with a standard
deviation of less than 10% in a group of 10 animals.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, PN-1,
(37)
, and PAI-1, potent inhibitors for t-PA
(38)
. We studied the expression of these serpins and t-PA
after cerebral ischemia in mice (Fig. 1
). After middle cerebral artery occlusion, total RNAs from both
ipsilateral and contralateral cortices were harvested. mRNA samples (1
µg) were reverse transcribed with poly-dT oligonucleotides. An
aliquot of cDNA libraries (1 µl from 20 µl) was amplified by PCR
with specific oligonucleotides for ß-actin. Then 1 µl of the same
cDNA libraries was amplified with sets of specific oligonucleotides. As
shown in Fig. 1
, all our PCR reactions showed the same level of
expression of the chosen housekeeping gene (ß-actin). Each set of the
oligonucleotides used gave us products of the expected size (252 bp for
t-PA; 322 bp for PAI-1; 224 bp for PN-1; 400 bp for NSP; and 539 bp for
ß-actin). To test the specificity of the PCR products, each was
cloned and sequenced. Furthermore, a PCR control using
ß-actin-specific primers was performed with a representative RNA
sample harvested from the cerebral cortices of mice to rule out any
genomic DNA contamination. The PAI-1 cDNA products obtained from both
ipsilateral and contralateral cortices from the 12 h sham-operated
mice failed to show any significant modification. In contrast, the
expression of the PAI-1 mRNAs was dramatically enhanced between 24 h and 3 days, whereas no significant modification of the expression
level of the mRNAs coding for PN-1, NSP, or t-PA was observed. By using
our cloned PAI-1-PCR product, in situ hybridization using
35S-PAI-1 antisense cRNA probe was performed.
Figure 2
B shows that the PAI-1 gene is weakly expressed in healthy cerebral
cortex of adult mice, as previously observed in the semiquantitative
RT-PCR experiments. Similarly, the expression pattern of PAI-1 was
similar to that described in Fig. 1
, with an overexpression of the
PAI-1 gene in the injured hemisphere (Fig. 2C, E
)
compared to the contralateral hemisphere (Fig. 2D
). Based on
our PCR experiments, this overexpression of PAI-1 occurred between
24 h and 3 days after cerebral ischemia (Fig. 2C, E
). Using an adjacent section immunostained with the
anti-glial fibrillary-associated protein (GFAP) antibody, we
established that the expression of PAI-1 is located in the region of
GFAP positive astrocytes (Fig. 2A
).
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Serpins and t-PA expression pattern in cultured astrocytes and
neurons: modulation by TGF-ß1
We further studied the cellular distribution of t-PA and serpins
(NSP, PN-1, and PAI-1) in both glial cultures and pure cultures of
cortical neurons. By using RT-PCR, we showed that t-PA mRNA was
expressed in both neurons and astrocytes. Astrocytes and neurons both
exhibited PN-1 and NSP, but the mRNA expression of PAI-1 was restricted
to the astrocytes (Fig. 3
). Although the expression of t-PA, PN-1, and NSP mRNAs was not
influenced by TGF-ß1 treatment in astrocytic and neuronal cultures,
an enhancement of the expression of PAI-1 mRNA after TGF-ß1 treatment
was observed in the astrocytes but not in pure neuronal cultures.
Immunocytochemistry shows an expression of PAI-1 in cultured astrocytes
but not in pure neuronal cultures (Fig. 4
).
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TGF-ß1-induced up-regulation of PAI-1 in astrocytes involves the
activation of an Smad3/Smad4-inducible element
PAI-1 expression in astrocytes subsequent to TGF-ß1 treatment
was studied at the transcriptional level. To quantify PAI-1
transcriptional activity that depends on TGF-ß1, we developed two
luciferase reporter genes driven either by the full-length PAI-1
promoter (PAI-luciferase) or the TGF-ß-responsive element recently
described (CAGA box) (30)
. This latter sequence
specifically mediates the TGF-ß signal through binding of the
Smad3/Smad4 complex. The luciferase activity was normalized with
respect to a control vector and directly reflects the level of the
PAI-1 transcription. TGF-ß1 increased luciferase activity by around
75% for the full-length promoter of PAI-1 and to up to 3.6-fold for
the TGF-ß-inducible element. Cotransfection of both reporter genes
with the TßR-IIEI dominant negative receptor (39)
totally prevented these responses (Fig. 5
). These data were confirmed by RT-PCR and showed that the up-regulation
of PAI-1 transcriptional activity in astrocytes occurred as early as
3 h after TGF-ß1 addition (Fig. 6
) and was maintained as long as TGF-ß1 was present in the incubating
bath. Moreover, overexpression of PAI-1 after 6 h of TGF-ß1
treatment was noted at the protein level by SDS-PAGE of extracellular
matrix-associated proteins, followed by Western blot analysis through
the use of an antibody raised against PAI-1 (Fig. 7
A). Similar experiments, performed after metabolic labeling
using 35S methionine/cysteine, confirmed that
TGF-ß1 treatment induced a neosynthesis of PAI-1 in astrocytes (Fig. 7B
).
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Overexpression of PAI-1 in astrocytes is induced by TGF-ß and
activin but not by BMP, GDNF, and NTN
Specific serine/threonine kinase type I receptors transduce the
intracellular signaling of various members of the TGF-ß family such
as BMPs, TGF-ßs, and activins (40)
. GDNF and NTN are
novel members of the TGF-ß family that transduce their signal through
activation of a common tyrosine kinase receptor, c-Ret. To test the
specificity of TGF-ß on the expression of PAI-1, we cotransfected
astrocytes with expression vectors encoding for constitutively
activated versions of the type I receptors (Fig. 8
C) (41)
in the presence of either the
full-length PAI-1 promoter or the TGF-ß-responsive element (CAGA box)
contained in the full-length PAI-1 promoter driving the luciferase
reporter gene. All of these experiments were normalized according to
the activity of a cotransfected control vector as described in
Materials and Methods. As shown in Fig. 8
, expression of ALK-4/T206D
and ALK-5/T204D led to transcriptional activation of the PAI-1
full-length promoter and of the TGF-ß-responsive element (CAGA box).
In contrast, expression of ALK-2/Q207D, ALK3/Q233D ,and ALK-6/Q204D did
not show any significant effect on the activation of the full-length
promoter, demonstrating that the PAI-1 promotor is activated by TGF-ß
and activin but not by BMP. We also tested the effect of both GDNF and
NTN on the transcriptional activity of the PAI-1 full-length promoter
and the CAGA box, with no significant activation (Fig. 9
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 22
, 42)
. Similarly, in
a model of focal permanent ischemia in mice, we confirmed an
overexpression of TGF-ß1 mRNAs after 24 h and up to 3 days
(8)
. Overall, these findings suggest that TGF-ß could
play a critical role in the neuropathological consequences of ischemia.
TGF-ß1 has been shown to protect central neurons against diverse
metabolic and excitotoxic challenges (17
, 21)
. However,
others have reported that under certain conditions TGF-ß1 was able to
potentiate glutamate-induced neuronal injury (43
44
45)
. We
previously investigated the influence of TGF-ß1 in apoptosis and
necrosis, two mechanisms involved in ischemic neuronal death. We showed
that TGF-ß1 exerts a beneficial effect against NMDA- but not against
AMPA- or kainate-induced necrosis. We extended this observation by
demonstrating that PAI-1, a serine protease inhibitor, expressed in
astrocytes and overexpressed after TGF-ß1 treatment, mediated the
neuroprotective effect of TGF-ß1 (31)
. These results
raised the question of the implication of serine proteases and their
inhibitors in the pathophysiology of ischemia.
Serine proteases have been involved in a variety of processes during
the development of the nervous system as well as in the physiology of
adult brain. For example, thrombin and plasminogen activators (PAs) are
known to modulate the neurite outgrowth in cell culture
(46)
. More recently a function for PAs in synaptic
plasticity and memory consolidation has been suggested, as plasminogen
activator (t-PA) mRNA is increased in the hippocampus during the
induction of long-term potentiation (LTP) (2)
and in the
cerebellum after the learning of a complex motor task
(47)
. Furthermore, membrane depolarization in PC12 cells
induces the release of t-PA (48)
, and mice deficient for
t-PA are resistant to neuronal loss after the intrahippocampal
injection of excitotoxins (3)
or ischemic insult
(4)
. These data suggest that serine proteases such as t-PA
are involved in the physiological processes and pathological mechanisms
that occur during neuronal degeneration. Serine proteases with a role
in the nervous system are thought to be regulated by serine protease
inhibitors, termed serpins. The expression pattern of serpins (PAI-1,
NSP, and PN-1) seems to demonstrate a restricted overexpression of
PAI-1 after permanent focal cerebral ischemia. An equilibrium between
serine proteases and their specific inhibitors (serpins) exists in many
tissues where these agents are involved in physiological processes
associated with inflammation or connective tissue turnover; in
contrast, no clear involvement of serpins has been proposed for either
physiological or pathological processes within the central nervous
system.
We have shown that both NSP and PN-1 are expressed by cortical neurons
and astrocytes in culture, as has been demonstrated for t-PA. In
contrast, the expression of PAI-1 mRNA and protein is clearly
restricted to astrocytes. Moreover, TGF-ß1 is able to induce an
up-regulation of PAI-1 in astrocytes without any effect on the
expression of the others serpins expressed in the CNS. The time course
of the expression of PAI-1 after middle cerebral artery occlusion in
mice matched the expression pattern of TGF-ß1 under the same
conditions (8)
, which might suggest that the
overexpression of PAI-1 after TGF-ß1 treatment, observed in
vitro, could reflect the in vivo process occurring
during cerebral ischemia.
The bone morphogenetic proteins BMP-2, BMP-4, BMP-6, and BMP-7
(49)
and their receptors, BMPR-IA, BMPR-IB, and BMPR-II
mRNAs, are constitutively expressed in the developing mouse CNS
(50)
and the adult rat brain (51)
. Moreover,
BMPs are involved in both morphogenesis and neurogenesis in the mouse
(52)
. BMPs are also able to inhibit the proliferation of
astrocytes and to induce their differentiation (53
, 54)
.
Messenger RNAs for activin and the activin receptors ActR-II, ActR-I,
and ActR-IB are found in the developing rat spinal cord and cortex
(51)
and the adult rat brain (55)
. Activin is
known to exert a neurotrophic effect on cultured hippocampal neurons
(56)
. In addition to these TGF-ß related cytokines, two
other members of the same family—the glial-derived neurotrophic factor
(14)
and neurturin (15)
—which are associated
with tyrosine kinase receptors, have recently been identified. These
two members have been characterized as neurotrophic cytokines able to
prevent neurodegeneration of dopaminergic neurons. In the present
study, we found that the expression of PAI-1 in astrocytes is enhanced
by TGF-ß and potentially by activin, but not by other members of the
family such as BMPs, GDNF, or NTN. Moreover, our results obtained with
the TGF-ß-responsive element (CAGA box) suggest that the TGF-ß
signaling pathway in astrocytes involves Smad3 and Smad4 complexes, as
previously characterized in peripheric tissues.
PAI-1 is a serpin that regulates the proteinase cascade initiated by
t-PA. In a previous study, an increased expression of PAI-1 has been
observed in the cerebrospinal fluid from patients with various
neurological diseases such as Alzheimer's disease, cerebral
infarction, CNS infection, and CNS neoplasia (57)
. More
recently, the serpins/t-PA axis has been shown to be involved in the
excitotoxic cascade induced after cerebral ischemia (3
, 4)
. Our present data suggest that the overexpression of TGF-ß1
that occurs after cerebral ischemia may specifically promote an
increased expression of PAI-1.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication January 11, 1999.
Revision received March 6, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 (1) collagen gene expression through a cis acting element that contains an Sp1-binding site. J. Biol. Chem. 269,14828-14834This article has been cited by other articles:
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  L. Fredriksson, M. Ehnman, C. Fieber, and U. Eriksson Structural Requirements for Activation of Latent Platelet-derived Growth Factor CC by Tissue Plasminogen Activator J. Biol. Chem., July 22, 2005; 280(29): 26856 - 26862. [Abstract] [Full Text] [PDF]
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S. Lesne, F. Docagne, C. Gabriel, G. Liot, D. K. Lahiri, L. Buee, L. Plawinski, A. Delacourte, E. T. MacKenzie, A. Buisson, et al. Transforming Growth Factor-beta 1 Potentiates Amyloid-beta Generation in Astrocytes and in Transgenic Mice J. Biol. Chem., May 9, 2003; 278(20): 18408 - 18418. [Abstract] [Full Text] [PDF]
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 N. Hosomi, J. Lucero, J. H. Heo, J. A. Koziol, B. R. Copeland, and G. J. del Zoppo Rapid Differential Endogenous Plasminogen Activator Expression After Acute Middle Cerebral Artery Occlusion Stroke, June 1, 2001; 32(6): 1341 - 1348. [Abstract] [Full Text] [PDF]
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