© 1999 American Heart Association, Inc.
Thrombosis |
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
From the Department of Neurosurgery (P.T., L.W., S.Y., M.H.W., B.V.Z.), University of Southern California, Los Angeles; the Neuroscience Center (N.S.), University of Colorado Health Sciences Center, Denver; the Division of Neurosurgery (J.G.M., B.V.Z.), Children’s Hospital Los Angeles, University of Southern California, Los Angeles; Scripps Research Institute (J.H.G.), La Jolla, Calif; and the Center for Transgene Technology and Gene Therapy (P.C.), Flanders, Leuven, Belgium.
Correspondence to Berislav V. Zlokovic, MD, PhD, 2025 Zonal Ave, RMR 506, Los Angeles, CA 90033. E-mail zlokovic{at}hsc.usc.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—Although the serine protease, tissue plasminogen activator (tPA), is approved by the US Food and Drug Administration for therapy to combat focal cerebral infarction, the basic concept of thrombolytic tPA therapy for stroke was challenged by recent studies that used genetically manipulated tPA-deficient (tPA-/-) mice, which suggested that tPA mediates ischemic neuronal damage. However, those studies were potentially flawed because the genotypes of tPA-/- and wild-type control mice were not entirely clear, and ischemic neuronal injury was evaluated in isolation of tPA effects on brain thrombosis. Using mice with appropriate genetic backgrounds and a middle cerebral artery occlusion stroke model with nonsiliconized thread, which does lead to microvascular thrombus formation, in the present study we determined the risk for cerebrovascular thrombosis and neuronal injury in tPA-/- and genetically matched tPA+/+ mice subjected to transient focal ischemia. Cerebrovascular fibrin deposition and the infarction volume were increased by 8.2- and 6.7-fold in tPA-/- versus tPA+/+ mice, respectively, and these variables were correlated with reduced cerebral blood flow up to 58% (P<0.05) and impaired motor neurological score by 70% (P<0.05). Our findings indicate that tPA deficiency exacerbates ischemia-induced cerebrovascular thrombosis and that endogenous tPA protects the brain from an ischemic insult, presumably through its thrombolytic action. In addition, our study emphasizes the importance of appropriate genetic controls in murine stroke research.
Key Words: tissue plasminogen activator • cerebrovascular events • fibrin • neuroprotection • ischemia
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The majority of acute ischemic strokes in humans are due to thrombotic or thromboembolic occlusions.1 2 3 This forms the rationale for use of thrombolytic agents in patients with acute ischemic stroke. Intravenous injection of the serine protease, tissue plasminogen activator (tPA), has been approved by the US Food and Drug Administration to combat focal cerebral infarction or ischemic stroke, and modest but significant results have been reported in stroke victims if tPA is administered within 3 to 6 hours of the insult.4 5 6
The neuroprotective effects of tPA were demonstrated in initial experimental stroke studies in rodents7 8 9 but were not confirmed in later studies in mice lacking tPA (tPA-/-) due to genetic engineering.10 Studies in tPA-/- versus wild-type mice raised the possibility that endogenous tPA mediates excitotoxin-induced neuronal degeneration11 and is responsible for ischemic neuronal damage.10 It has been suggested that these neurotoxic effects of tPA are independent of its desirable thrombolytic action.10 These findings, although in contradiction with previously suggested physiological roles of tPA in brain development,12 learning, memory,13 and long-term potentiation,14 15 have challenged the concept of tPA lytic therapy for stroke. Whether tPA treatment for ischemic stroke may be neurotoxic has become a controversial issue in the treatment of stroke patients.
The work arguing for an ischemia-induced neurotoxic role of tPA has, however, compared ischemic brain injury in tPA-/- mice versus wild-type mice that were on different genetic backgrounds. In addition, ischemic neuronal injury was evaluated in isolation of tPA’s beneficial effect on brain thrombosis.10 Because genetic backgrounds16 17 18 19 20 and thrombosis21 22 significantly affect outcome in murine models of ischemia and neuronal damage, we examined the risk for cerebrovascular thrombosis and neuronal injury in tPA-/- and genetically matched tPA+/+ mice subjected to focal stroke. Our findings presented here suggest that tPA deficiency augments harmful cerebrovascular fibrin deposition that correlates with enhanced brain injury, thus supporting the view that tPA protects the brain from an ischemic insult through its thrombolytic action.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animals
Studies were performed in tPA-/- and tPA+/+ wild-type mice, both on matched mixed 129/Sv and C57BL/6 genetic backgrounds. The littermates of both lines were bred independently to mice with which they did not share a grandmother in common and were back-crossed every 8 to 10 generations to generate heterozygotes, thus excluding major genetic drift between both lines. The tPA-/- animals were routinely screened by tail bleeds to verify their tPA deficiency. The tPA+/+ C57BL/6 wild-type mice were used to control for strain-related differences in susceptibility to stroke. Male mice weighing between 22 and 27 g were subjected to a modified middle cerebral artery (MCA) occlusion technique23 as described below.
General Preparation
Mice were initially anesthetized with metofane and
maintained with 50 mg/kg IP pentobarbital. Atropine methyl nitrate
(0.18 mg/kg IP) was given as premedication to prevent airway
obstruction by mucus formation. Animals were allowed to breath
spontaneously. Rectal temperature was maintained at 37±1°C by a
thermostatically regulated heating pad. The right femoral artery was
cannulated with a PE-10 catheter for continuous monitoring of blood
pressure and blood analysis. All procedures were
done in accordance with the Animal Care guidelines at the University of
Southern California and approved by the National Institutes of Health.
Stroke Model
A modified intravascular MCA occlusion technique23
with a nonsiliconized, uncoated, 6-0 8-mm-long prolene suture was used
in mice weighing 22 to 27 g versus the commonly used 10- to
12-mm-long nylon filament coated with silicone that does not have
thrombogenic properties.10 In brief, under the operating
microscope the right carotid complex was exposed through a ventral
midline incision, and the external carotid artery and its branches
ligated. Through a transverse incision in the artery, the suture was
introduced into the external carotid artery lumen and gently advanced
into the internal carotid artery to occlude the MCA at its origin from
the circle of Willis. The MCA occlusion lasted for 3 hours and was
followed by 24 hours of reperfusion.
Blood Flow and Head Temperature Measurements
Cortical cerebral blood flow (CBF) was monitored by laser
Doppler flowmetry (LDF) and a tissue perfusion monitor
(Transonic BLF21). Animals were placed in a
stereotactic head frame, and readings were obtained 2
mm posterior to the bregma, both 3 and 6 mm to each side of the
midline, by using a stereotactic micromanipulator and
keeping the angle of the probe perpendicular to the cortical surface.
LDF probes (0.8-mm diameter) positioned on the cortical surface were
connected to a tissue perfusion monitor (Transonic BLF21). The
procedure was considered to be technically successful when a 
50%
reduction in relative CBF was observed immediately after the placement
of the occluding suture.17 Animals with inadequate blood
flow decrease were excluded from the study. These exclusion criteria
yielded the levels of ischemia sufficient to render
consistent infarct volumes after
2,3,5,-triphenyltetrazolium (TTC) staining.
Head temperature was monitored with a 36-gauge thermocouple temperature
probe in the temporalis muscle connected to a digital
thermometer/thermoregulator (model 9000, Omega).
Neurological Deficits
Neurological examinations were performed at 24 hours after
reperfusion. The neurological findings were scored on a 5-point
modified scale: no neurological deficit (0); failure to extend left
forepaw fully; (1) turning to left; (2) circling to left; (3) unable to
walk spontaneously; and (4) stroke-related death
(5).24
Blood Analysis
Arterial blood gases (pH,
PaO2, PaCO2) were measured
before and during MCA occlusion by using an ABL 30 acid-base
analyzer (Radiometer).
Measurement of Volume of Injury
The area of injury was delineated by incubation of unfixed 1-mm
coronal brain slices in 2% TTC in phosphate buffer (pH 7.4). Serial
coronal sections were displayed on a digitizing video screen equipped
with an imaging system (Jandel Scientific). The volume of injury was
calculated by summing the affected areas from each coronal section and
multiplying by the thickness of each section. Brain infarction and
edema were calculated using the Swanson correction.23
Histopathology
Detection of fibrin by immunostaining was
performed on brain tissue sections previously treated with TTC. Tissue
fixed in 10% buffer formalin was processed, and 7-µm-thick, paraffin
coronal sections from each block were cut and stained. Fibrin was
localized using a grading scale25 : 1, fibrin deposition
limited to intravascular space; 2, fibrin deposition in the
intravascular lumen and the perivascular space; and 3, fibrin lattices
in the extravascular or parenchymal tissue only. For
immunostaining, anti-mouse fibrin II antibody
(NYB-T2G1, Accurate Chemical Science Corp)26 (1:1000
dilution) was used. Routine control sections included deletion of
primary antibody, deletion of secondary antibody, and the use of an
irrelevant primary antibody. All analyses assessed visually
were performed by 1 observer blinded to the specimen source or
timing.
Detection of Fibrin in Brain Tissue Sections by Quantitative
Western Blot
In brief, after being stained with TTC, a 1-mm section of brain
tissue was divided into contralateral and ipsilateral hemispheres.
Other organs (heart, lung, liver, etc) were also collected. Tissue was
homogenized in 10 mmol/L sodium phosphate buffer, pH
7.5, 0.1 mol/L 
-amino-n-caproic acid, 5 mmol/L
trisodium EDTA, 10 U aprotinin/mL, 10 U heparin/mL, and 2 mmol/L
PMSF. The homogenate was agitated for 14 hours at 4°C,
and the particulate material was sedimented by
centrifugation at 10 000g for 10 minutes,
resuspended in extraction buffer without PMSF, sedimented again, and
finally dispersed in 3 mol/L urea. The suspension was agitated for 2
hours at 37°C, vigorously vortexed, and centrifuged at
14 000g for 15 minutes. The supernatant was aspirated and
the sediment dissolved at 65°C in reducing SDS buffer, subjected to
SDS–polyacrylamide gel electrophoresis (8%), and transferred
to a polyvinylidine difluoride membrane (Immobilon-P; Millipore
Corp) by electroblotting.26 Fibrin was visualized
with anti-mouse fibrin II antibody (given above) and an enhanced
chemiluminescence system (Amersham Corp). Fibrin standards were
prepared by clotting a known amount of murine fibrinogen (Sigma
Chemical Co) with an excess of thrombin in the absence of calcium. The
films were scanned with a Hoefer GS 300 scanning densitometer
interfaced to an IBM personal computer with a DT 2805 analog and
digital system (Data Translation), and data were converted into
micrograms of fibrin per 0.1 gram of tissue.
Statistics
Physiological variables, infarction, and
edema volumes were compared between groups by Student’s t
test and ANOVA. Nonparametric data (neurological outcome
scores) were subjected to the Kruskal-Wallis test. A value of
P<0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
First, we examined the anatomy of the circle of Willis in mixed tPA-/- and tPA+/+ 129/Sv and C57BL/6 strains and C57BL/6 mice, as the cerebral vascular pattern may influence the susceptibility of mice to cerebral ischemia.18 19 20 Figure 1A
illustrates the complete circle of
Willis in a mixed 129/Sv and C57BL/6 tPA-/- mouse after intracardiac
Evans blue perfusion; the same vascular pattern was found in a mixed
tPA+/+ control mouse (not shown). The C57BL/6 mouse strain has a
well-developed forebrain circulation including the MCA, and the
anterior circulation is similar to that of the mixed 129/Sv and C57BL/6
strain.18 19 20 However, in contrast to the mixed strain,
the C57BL/6 mouse has dysgenesis of the posterior cerebral
communicating artery, and contributions of the posterior circulation to
the forebrain anterior and middle cerebral circulations are negligible
in this strain.18 19 20
|
We used 8-mm-long, 6-0, uncoated, nonsiliconized prolene sutures that
may have different occluding and thrombogenic properties in comparison
with the more-often-used 10- to 12-mm-long nylon filament coated with
silicone, which does not lead to thrombus formation.10 The
position of thread in the MCA is shown in Figure 1B
.
In all animals, rectal temperature was maintained at 37±1°C. The
blood pressure, gases, and the pH before and after 3 hours of MCA
occlusion were within the physiological range in
all studied groups. After 3 hours of occlusion, animals were allowed a
24-hour reperfusion period before they were killed for
analysis. Brain injury at 24 hours in tPA-/- mice was
significant and involved the lateral, ventromedial, and dorsolateral
cortex and the lateral and medial striatum, in contrast to limited
injury in the tPA+/+ mixed control strain (Figure 1C). On the
other hand, the injury in C57BL/6 mice was significantly greater than
in both tPA-/- and tPA+/+ mice on 129/Sv and C57BL/6 backgrounds,
reflecting significant genetic differences between the 2 strains
(Figure 1C). Figure 2A illustrates
that the volumes of brain infarction and edema were significantly
increased, by 6.7- and 2.3-fold, respectively, in tPA-/- mice versus
wild-type tPA+/+ mixed strain control mice. This correlated with
impaired CBF in tPA-/- versus tPA+/+ mice on the same genetic
background, both during the MCA occlusion by 39% (P<0.05
by ANOVA) and during reperfusion with an average decline of 58% within
the first hour (P<0.05 by ANOVA; Figure 2B).
Neurological deficit at 24 hours of reperfusion was significantly
higher in tPA-/- mice versus genetically matched tPA+/+ mice, ie, 2.4
versus 1.4 (P<0.05 by Kruskal-Wallis test). The size of
brain infarcts in C57BL/6 mice was 9.7-fold larger than in mixed 129/Sv
and C57BL/6 mice that served as tPA+/+ wild-type matched controls,
whereas there was no significant difference in the edema volume between
the 2 strains (Figure 2A). The CBF reduction during occlusion
and the recovery during reperfusion phase in C57BL/6 mice were not
significantly different from control tPA+/+ 129/Sv and C57BL/6
mixed-strain mice. The neurological score, however, in C57BL/6 mice was
3.2; it is notable that this value was significantly higher
(P<0.05 by Kruskal-Wallis test) than in both tPA+/+ and
tPA-/- mixed strain mice, emphasizing that mouse strain is an
important variable in murine stroke models.
|
Figure 3 shows fibrin deposits in
microvessels in the ischemic lesioned hemisphere in tPA-/-
mice. The immunostaining for fibrin was performed after
TTC staining. Several small arterioles, venules, and capillaries
contained intraluminal fibrin deposits (Figure 3A) of
grade 1 on the scale for fibrin localization in an MCA
model.25 Extravascular deposition of fibrin grade 2
was also found, suggesting breakdown of the blood-brain barrier (Figure 3A). Figure 3B shows several capillaries in the
ischemic hemisphere positive for fibrin, in contrast to
insignificant fibrin staining in a matched region of the contralateral
hemisphere (Figure 3C). Figures 3D, 3E, and 3F illustrate
fibrin deposits in a large pial venule and parenchymal capillaries,
intraluminal and perivasular deposits around a small microvessel, and
fibrin deposits within capillaries, respectively. Several vessels
contained entrapped blood cells (eg, see venules in Figure 3A or
the small microvessel in Figure 3E), possibly reflecting
polymorphonuclear response (indicated by positive staining for
polymorphonuclear markers; not shown) as reported
previously.27
|
Fibrin deposits and fibrin-rich thrombi were found occasionally
after 24 hours of reperfusion in tPA+/+ wild-type 129/Sv and C57BL/6
mice in some venules, but most capillaries and other microvessels were
negative for fibrin (Figures 3G and 3H). Quantitative Western
blot analysis for fibrin on 1-mm-thick TTC brain sections
confirmed the immunohistological observations by
demonstrating significant fibrin deposition in ischemic brain
areas in tPA-/- mice versus barely detectable levels of fibrin in
tPA+/+ wild-type control mixed-strain mice (Figure 4C). The relative increase in the amount
of fibrin in brain section IV (the level of the optic chiasm) was
8.2-fold based on Western blot analysis and in sections III
and V, between 5- and 6-fold. In the absence of ischemic
challenge, there were no fibrin deposits in brain under basal
conditions in any strain of either tPA-/- or tPA+/+ mice. Fibrin
deposition in the ischemic hemisphere in tPA+/+ C57BL/6 mice
subjected to ischemia was barely detectable (not shown) and was
similar to that in tPA+/+ 129/Sv and C57BL/6 mixed-strain control mice,
indicating that the fibrin level was not related to the difference in
injury volumes between the 2 strains. No genotypic differences in
endothelial lining, such as denudation, that could
precipitate fibrin deposition were observed, and the
immunoblot signal for fibrin deposition in control
nonischemic brain was undetectable in all groups of mice (data
not shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
This study in tPA-/- and wild-type tPA+/+ mice on a matched genetic 129/Sv and C57BL/6 mixed background indicates that tPA deficiency precipitates and markedly augments ischemia-induced cerebrovascular fibrin deposition and microvascular thrombotic obstructions. This in turn correlates with enhanced brain infarction and edema volumes, reduced CBF during MCA occlusion, and the reperfusion phase after the ischemic insult, and impaired motor neurological score 24 hours after focal cerebral ischemia in tPA-/- mice. The study strongly suggests that tPA protects the brain from an ischemic insult through its desirable thrombolytic action.
Cerebral protection from tPA has been shown in earlier stroke studies7 8 9 that led to clinical trials with tPA.4 5 6 Unwanted side effects of tPA treatment for stroke were also noted, including a higher rate of intracerebral hemorrhage,28 downstream displacement of thrombolysed material, breakdown of the blood-brain barrier, and reperfusion injury.29 However, direct neurotoxic effects from tPA have not been reported until recently. Recent studies in tPA-/- 129/Sv and C57BL/6 mixed-strain mice compared with C57BL/6 mice that served as a wild-type tPA+/+ control suggested that tPA mediates neuronal damage after cerebral ischemia.10 These studies raised a question whether tPA should be discontinued as an antistroke agent because of its neurotoxicity,10 or alternatively, whether clinical tPA trials should better define the inclusion/exclusion criteria for tPA treatment in the stroke population if tPA does not harm neurons.1 2 3
The present study compared 2 wild-type tPA+/+ mouse strains, the C57BL/6 strain and the 129/Sv and C57BL/6 mixed strain, and tPA-/- versus tPA+/+ mice on matched 129/Sv and C57BL/6 mixed genetic background. Because the volume of ischemic brain injury with the vascular occlusion method is inversely proportional to the length of thread inserted into the MCA,17 we used shorter threads to increase the sensitivity of measurements. In contrast to commonly used siliconized nylon sutures, which do not lead to thrombus formation,10 we used nonsiliconized thread that might have different thrombogenic properties30 and does lead to microvascular thrombus formation. Our data confirmed that the wild-type C57BL/6 strain is significantly more susceptible to cerebral ischemia than other strains for reasons that are not well understood.17 18 19 20 For example, infarcts in C57BL/6 mice are 7-fold larger than in 129J mice17 and 9.6-fold larger than in mixed 129/Sv and C57BL/6 mice as shown in the present study, and there is also significantly higher susceptibility to ischemia in C57BL/6 mice compared with 129/Sv mice.18 19 20 It is possible that the use of the highly susceptible C57BL/6 mouse to serve as a control for the effects of ischemia in mismatched tPA-deficient mouse strains, eg, the mixed 129/Sv and C57Bl/6 strain in a previous study,10 could in addition to the differences in the ischemia models, eg, without thrombosis10 versus with thrombosis and cerebrovascular fibrin deposition (present study), account for differences in the results and data interpretation between the 2 studies.
Mice with combined tPA and urokinase deficiencies spontaneously deposit fibrin in several organs associated with ischemic necrosis.31 32 The singly deficient mice, either urokinase PA-/- or tPA-/-, do not develop spontaneous fibrin deposits under basal conditions, but they are significantly more susceptible to development of venous or arterial thrombosis if exposed to provocative stimuli (eg, endotoxin) or subjected to injury of the peripheral arteries.31 33 Our present data concur with these studies by demonstrating increased brain fibrin deposition, enhanced brain injury, and reduced CBF in tPA-deficient mice versus genetically matched tPA+/+ mice challenged by an ischemic insult. It is possible that small differences in the CBF within the critical range of 0% to 30% of baseline during the 3 hours of MCA occlusion may have a major influence on neuropathological outcome in tPA-/- versus genetically matched tPA+/+ mice. On the other hand, under basal conditions, there was no detectable fibrin deposits in brains of either tPA-/- or tPA+/+ mice. Because the anatomy of the cerebral vascular pattern in tPA-/- versus genetically matched tPA+/+ mice was the same and genotypic differences in endothelial lining, such as denudation, that could precipitate fibrin deposition were not observed, it is most likely that enhanced cerebrovascular fibrin deposition and brain injury in tPA-/- mice are related to the lack of fibrinolytic tPA action. However, one cannot rule out that other potentially important hemostatic differences in brain endothelial cells may exist between tPA-/- and tPA+/+ mice that can also contribute to alterations in the coagulation profile within the brain microcirculation in tPA-/- versus tPA+/+ mice.
The 2 tPA+/+ wild-type strains, namely, mixed 129/Sv and C57BL/6 mice and C57BL/6 mice, did not develop significant cerebrovascular fibrin deposits when subjected to focal ischemic stroke, and only occasionally did some venules and/or capillaries display moderate positive intraluminal fibrin staining. This may indicate that endogenous tPA in brain capillaries and/or in the circulation of these mice is sufficient to maintain significant thrombolytic activity. The 2 control tPA+/+ strains did not exhibit significant differences in either CBF during occlusion and the reperfusion phase or in the edema volume or pattern of forebrain cerebral circulation. Thus, it is most likely that as-yet-unidentified genotypic factors and possibly poor communication between posterior and forebrain circulations in C57BL/6 mice17 18 19 20 compared with 129/Sv and C57BL/6 mixed mice that exhibit a normal circulatory pattern may be responsible for the significantly larger infarction volumes in the gray matter and higher neurological scores in C57BL/6 mice versus the 129/Sv and C57BL/6 mixed strain or other mouse strains.17 18 19 20
Fibrin deposition in ischemic brain can be precipitated by procoagulant transformation of brain microvascular endothelium,21 22 23 25 formation of a platelet plug, or leukocyte-endothelial interactions during reperfusion.27 Previous studies have established a link between ischemic brain injury and reduced antithrombotic mechanisms in brain microcirculation in the presence of major stroke risk factors.23 34 35 In humans, the antithrombotic factor protein C is protective in ischemic stroke.36 The present study confirms that a deficiency of tPA favors fibrin deposition in brains challenged by a thrombotic ischemic insult, which in turn enhances ischemic brain injury. These findings provide direct evidence for a causal relationship between reduced fibrinolytic activity and ischemic neuronal damage, suggesting that endogenous tPA promotes desirable thrombolytic and protective effects in cerebral ischemia, and that factors other than tPA must be responsible for ischemic neuronal damage. In addition, the present study emphasizes the importance of appropriate genetic controls in murine stroke research.
|
Acknowledgments |
|---|
This work was supported by National Institutes of Health grant HL63290 to B.V.Z.
|
Footnotes |
|---|
Preliminary findings were reported to the Society of Neuroscience, Los Angeles, Calif, at its 28th Annual Meeting, November 7–12, 1998, and published in abstract form (p 1256).
Received March 16, 1999; accepted May 19, 1999.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. del Zoppo GJ. tPA: a neuron buster, too? Nat Med. 1998;4:148–150.[Medline] [Order article via Infotrieve]
2. Zivin JA. Recent advances in stroke therapy. Western J Med. 1998;168:261–262.[Medline] [Order article via Infotrieve]
3.
Zivin JA. Factors determining the therapeutic window
for stroke. Neurology. 1998;50:599–603.
4.
The National Institutes of Neurological Disorders, and
Stroke rtPA Stroke Study Group. Tissue plasminogen
activator for acute ischemic stroke. N
Engl J Med. 1995;333:1581–1587.
5.
Hacke W, Kaste M, Fieschi C, Toni D, Lesaffre E, von
Kummer R, Boysen G, Bluhmki E, Hoxter G, Mahagne MH, Hennerici M.
Intravenous thrombolysis with recombinant
tissue plasminogen activator for acute
hemispheric stroke: the European Cooperative Acute Stroke Study
(ECASS). JAMA. 1995;274:1017–1025.
6.
del Zoppo GJ, Higashida RT, Furlan AJ, Pessin MS,
Rowley HA, Gent M. PROACT: a phase II randomized trial of recombinant
pro-urokinase by direct arterial delivery in acute middle
cerebral artery stroke. Stroke. 1998;29:4–11.
7.
Zivin JA, Fisher M, DeGirolami U, Hemenway CC, Stashak
JA. Tissue plasminogen activator reduces
neurological damage after cerebral embolism. Science. 1985;230:1289–1292.
8. Papadopoulos SM, Chandler WF, Salamat MS, Topol EJ, Sackellares JC. Recombinant human tissue-type plasminogen activator therapy in acute thromboembolic stroke. J Neurosurg. 1987;67:394–398.[Medline] [Order article via Infotrieve]
9. Overgaard K, Sereghy T, Pedersen H. Effect of delayed thrombolysis with rt-tPA in a rat embolic stroke model. J Cereb Blood Flow Metab. 1993;14:472–477.
10. 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]
11. 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]
12. Friedman GC, Seeds NW. Tissue plasminogen activator expression in the embryonic nervous system. Dev Brain Res. 1994;81:41–49.[Medline] [Order article via Infotrieve]
13.
Seeds NW, Williams BL, Bickford PC. Tissue
plasminogen activator induction in Purkinje
neurons after cerebellar motor learning. Science. 1995;270:1992–1994.
14. 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. 1994;361:453–457.
15.
Schauwecker PE, Steward O. Genetic determinants of
susceptibility to excitotoxic cell death: implications for gene
targeting approaches. Proc Natl Acad Sci U S A. 1997;94:4103–4108.
16.
Huang YY, Bach ME, Lipp HP, Zhuo M, Wolfer DP, Hawkins
RD, Schoonjans L, Kandel ER, Godfraind JM, Mulligan R, Collen D,
Carmeliet P. Mice lacking the gene encoding tissue-type
plasminogen activator show a selective
interference with late-phase long-term potentiation in both Schaffer
collateral and mossy fiber pathways. Proc Natl Acad Sci
U S A. 1996;93:8699–8704.
17. Connolly ES, Winfree CJ, Stern DM, Solomon RA, Pinsky DJ. Procedural and strain-related variables significantly affect outcome in a murine model of focal cerebral ischemia. Neurosurgery. 1996;38:523–532.[Medline] [Order article via Infotrieve]
18. Yang G, Kitagawa K, Matsushita K, Mabuchi T, Yagita Y, Yanagihara T, Matsumoto M. C57BL/6 strain is most susceptible to cerebral ischemia following bilateral common carotid occlusion among seven mouse strains: selective neuronal death in the murine transient forebrain ischemia. Brain Res. 1997;752:209–218.[Medline] [Order article via Infotrieve]
19.
Fujii M, Hara H, Meng W, Vonsattel JP, Huang Z,
Moskowitz MA. Strain-related differences in susceptibility to transient
forebrain ischemia in SV-129 and C57black/6 mice.
Stroke. 1997;28:1805–1811.
20. Terashima T, Namura S, Hoshimaru M, Uemura Y, Kikuchi H, Hashimoto N. Consistent injury in the striatum of C57BL/6 mice after transient bilateral common carotid artery occlusion. Neurosurgery. 1998;43:900–908.[Medline] [Order article via Infotrieve]
21. del Zoppo GJ. Microvascular changes during cerebral ischemia and reperfusion. Cerebrovasc Brain Metab Rev. 1994;6:47–96.[Medline] [Order article via Infotrieve]
22. Zlokovic BV, Wang L, Schreiber SS, Fisher M. Haemostatic functions of the blood-brain barrier: possible implications in the pathogenesis of stroke. In: Greenwood J, Begley DJ, Segal MB, eds. New Concepts of the Blood-Brain Barrier. New York, NY: Plenum Press; 1995:141–145.
23. Wang L, Kittaka M, Sun N, Schreiber SS, Zlokovic BV. Chronic nicotine treatment enhances focal ischemic brain injury and depletes brain capillary tissue plasminogen activator in rats. J Cereb Blood Flow Metab. 1997;17:136–146.[Medline] [Order article via Infotrieve]
24. Hara H, Huang PL, Panahian N, Fishman MC, Moskowitz MA. Reduced isoform of nitric oxide synthase after transient MCA occlusion. J Cereb Blood Flow Metab. 1996;16:605–611.[Medline] [Order article via Infotrieve]
25. Okada Y, Copeland BR, Fitridge R, Koziol JA, del Zoppo GJ. Fibrin contributes to microvascular obstructions and parenchymal changes during early focal cerebral ischemia and reperfusion. Stroke. 1994;25:1847–1853.[Abstract]
26. Weiler-Guettler H, Christie PD, Beeler D, Healy AM, Hancock WW, Rayburn H, Edelberg JM, Rosenberg RD. A targeted point mutation in thrombomodulin generates viable mice with a prethrombotic state. J Clin Invest. 1998;101:1983–1991.[Medline] [Order article via Infotrieve]
27. Palabrica T, Lobb R, Furie BC, Aronovitz M, Benjamin C, Hsu Y, Sajar SA, Furie B. Leukocyte accumulation promoting fibrin deposition is mediated in vivo by P-selectin on adherent platelets. Nature. 1992;359:848–851.[Medline] [Order article via Infotrieve]
28.
The NINDS t-PA Stroke Study Group.
Intracerebral hemorrhage after
intravenous t-PA therapy for ischemic stroke.
Stroke. 1997;28:2109–2118.
29. Busch E, Kruger K, Allegrini PR, Kerskens CM, Gyngell ML, Hoehn-Berlage M, Hossmann KA. Reperfusion after thrombolytic therapy of embolic stroke in the rat: magnetic resonance and biochemical imaging. J Cereb Blood Flow Metab. 1998;18:407–418.[Medline] [Order article via Infotrieve]
30.
Belayev L, Alonso OF, Busto R, Zhao W, Ginsberg MD.
Middle cerebral artery occlusion in the rat by intraluminal suture:
neurological and pathological evaluation of an improved model.
Stroke. 1996;27:1616–1623.
31. Carmeliet P, Schoonjans L, Kieckens L, Ream B, Degen J, Bronson R, De Vos R, van den Oord JJ, Collen D, Mulligan RC. Physiological consequences of loss of plasminogen activator gene function in mice. Nature. 1994;368:419–424.[Medline] [Order article via Infotrieve]
32.
Bugge TH, Flick MJ, Daugherty CC, Degen JL.
Plasminogen deficiency causes severe thrombosis but is
compatible with development and reproduction. Genes
Dev. 1995;9:794–807.
33. Carmeliet P, Collen D. Gene manipulation and transfer of the plasminogen system and coagulation system in mice. Semin Thromb Haemost.. 1996;22:525–542.[Medline] [Order article via Infotrieve]
34.
Kittaka M, Wang L, Sun N, Schreiber SS, Seeds NW,
Fisher M, Zlokovic BV. Brain capillary tissue plasminogen
activator in a diabetes stroke model. Stroke. 1996;27:712–719.
35. Zlokovic BV. Antithrombotic, procoagulant, and fibrinolytic mechanisms in cerebral circulation: implications for brain injury and protection. Neurosurg Focus. 1997;2():1–13.
36. Folsom AR, Rosamond WD, Shahar E, Cooper LS, Aleksic N, Mieto ML, Rasmussen L, Wu KK. Prospective study of markers of hemostatic function, inflammation, and endothelial function with risk of ischemic stroke. Circulation. 1998;28(suppl I):I-207. Abstract.
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]
|
|
|
|
 |
|
 S. Inaba, M. Iwai, Y. Tomono, I. Senba, M. Furuno, H. Kanno, H. Okayama, M. Mogi, J. Higaki, and M. Horiuchi Exaggeration of Focal Cerebral Ischemia in Transgenic Mice Carrying Human Renin and Human Angiotensinogen Genes Stroke, February 1, 2009; 40(2): 597 - 603. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Iwanami, M. Mogi, J.-M. Li, K. Tsukuda, L.-J. Min, A. Sakata, T. Fujita, M. Iwai, and M. Horiuchi Deletion of Angiotensin II Type 2 Receptor Attenuates Protective Effects of Bone Marrow Stromal Cell Treatment on Ischemia-Reperfusion Brain Injury in Mice * Supplemental Materials and Methods Stroke, September 1, 2008; 39(9): 2554 - 2559. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Adhami, D. Yu, W. Yin, A. Schloemer, K. A. Burns, G. Liao, J. L. Degen, J. Chen, and C.-Y. Kuan Deleterious Effects of Plasminogen Activators in Neonatal Cerebral Hypoxia-Ischemia Am. J. Pathol., June 1, 2008; 172(6): 1704 - 1716. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Orset, R. Macrez, A. R. Young, D. Panthou, E. Angles-Cano, E. Maubert, V. Agin, and D. Vivien Mouse Model of In Situ Thromboembolic Stroke and Reperfusion Stroke, October 1, 2007; 38(10): 2771 - 2778. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Paul, S. Strickland, and J. P. Melchor Fibrin deposition accelerates neurovascular damage and neuroinflammation in mouse models of Alzheimer's disease J. Exp. Med., August 6, 2007; 204(8): 1999 - 2008. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. Hafer-Macko, F. M. Ivey, J. D. Sorkin, and R. F. Macko Microvascular tissue plasminogen activator is reduced in diabetic neuropathy Neurology, July 17, 2007; 69(3): 268 - 274. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Atkinson, H. Zhu, F. Qiao, J. C. Varela, J. Yu, H. Song, M. S. Kindy, and S. Tomlinson Complement-Dependent P-Selectin Expression and Injury following Ischemic Stroke J. Immunol., November 15, 2006; 177(10): 7266 - 7274. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-H. Liang and D.-M. Chuang Differential Roles of Glycogen Synthase Kinase-3 Isoforms in the Regulation of Transcriptional Activation J. Biol. Chem., October 13, 2006; 281(41): 30479 - 30484. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Wilczak, J. W. Elting, D. Chesik, I. P. Kema, and J. De Keyser Intravenous Tissue Plasminogen Activator in Patients With Stroke Increases the Bioavailability of Insulin-Like Growth Factor-1 Stroke, September 1, 2006; 37(9): 2368 - 2371. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J.T.H. Roelofs, K. M.A. Rouschop, J. C. Leemans, N. Claessen, A. M. de Boer, W. M. Frederiks, H.R. Lijnen, J. J. Weening, and S. Florquin Tissue-Type Plasminogen Activator Modulates Inflammatory Responses and Renal Function in Ischemia Reperfusion Injury J. Am. Soc. Nephrol., January 1, 2006; 17(1): 131 - 140. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Jood, P. Ladenvall, A. Tjarnlund-Wolf, C. Ladenvall, M. Andersson, S. Nilsson, C. Blomstrand, and C. Jern Fibrinolytic Gene Polymorphism and Ischemic Stroke Stroke, October 1, 2005; 36(10): 2077 - 2081. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Reddrop, R. X. Moldrich, P. M. Beart, M. Farso, G. T. Liberatore, D. W. Howells, K.-U. Petersen, W.-D. Schleuning, and R. L. Medcalf Vampire Bat Salivary Plasminogen Activator (Desmoteplase) Inhibits Tissue-Type Plasminogen Activator-Induced Potentiation of Excitotoxic Injury Stroke, June 1, 2005; 36(6): 1241 - 1246. [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]
|
|
|
|
|
|
D.N. Atochin, J.C. Murciano, Y. Gursoy-Ozdemir, T. Krasik, F. Noda, C. Ayata, A.K. Dunn, M.A. Moskowitz, P.L. Huang, and V.R. Muzykantov Mouse Model of Microembolic Stroke and Reperfusion Stroke, September 1, 2004; 35(9): 2177 - 2182. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Iwai, H.-W. Liu, R. Chen, A. Ide, S. Okamoto, R. Hata, M. Sakanaka, T. Shiuchi, and M. Horiuchi Possible Inhibition of Focal Cerebral Ischemia by Angiotensin II Type 2 Receptor Stimulation Circulation, August 17, 2004; 110(7): 843 - 848. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. Melchor, R. Pawlak, and S. Strickland The Tissue Plasminogen Activator-Plasminogen Proteolytic Cascade Accelerates Amyloid-{beta} (A{beta}) Degradation and Inhibits A{beta}-Induced Neurodegeneration J. Neurosci., October 1, 2003; 23(26): 8867 - 8871. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Ellsworth, R. Garcia, J. Yu, and M. S. Kindy Fibroblast Growth Factor-18 Reduced Infarct Volumes and Behavioral Deficits After Transient Occlusion of the Middle Cerebral Artery in Rats Stroke, June 1, 2003; 34(6): 1507 - 1512. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Liu, H. Guo, J. H. Griffin, J. A. Fernandez, and B. V. Zlokovic Protein S Confers Neuronal Protection During Ischemic/Hypoxic Injury in Mice Circulation, April 8, 2003; 107(13): 1791 - 1796. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Singh, K.G. Burnand, M. Collins;, A. Luttun, D. Collen, B. Boelhouwer;, and A. Smith Failure of Thrombus to Resolve in Urokinase-Type Plasminogen Activator Gene-Knockout Mice: Rescue by Normal Bone Marrow-Derived Cells Circulation, February 18, 2003; 107(6): 869 - 875. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. T. Liberatore, A. Samson, C. Bladin, W.-D. Schleuning, and R. L. Medcalf Vampire Bat Salivary Plasminogen Activator (Desmoteplase): A Unique Fibrinolytic Enzyme That Does Not Promote Neurodegeneration Stroke, February 1, 2003; 34(2): 537 - 543. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Niessen, T. Hilger, M. Hoehn, and K.-A. Hossmann Thrombolytic Treatment of Clot Embolism in Rat: Comparison of Intra-arterial and Intravenous Application of Recombinant Tissue Plasminogen Activator Stroke, December 1, 2002; 33(12): 2999 - 3005. [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]
|
|
|
|
|
|
E. Kilic, M. Bahr, and D. M. Hermann Effects of Recombinant Tissue Plasminogen Activator After Intraluminal Thread Occlusion in Mice: Role of Hemodynamic Alterations Stroke, November 1, 2001; 32(11): 2641 - 2647. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Shibata, S. R. Kumar, A. Amar, J. A. Fernandez, F. Hofman, J. H. Griffin, and B. V. Zlokovic Anti-Inflammatory, Antithrombotic, and Neuroprotective Effects of Activated Protein C in a Murine Model of Focal Ischemic Stroke Circulation, April 3, 2001; 103(13): 1799 - 1805. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Yepes, M. Sandkvist, M. K. K. Wong, T. A. Coleman, E. Smith, S. L. Cohan, and D. A. Lawrence Neuroserpin reduces cerebral infarct volume and protects neurons from ischemia-induced apoptosis Blood, July 15, 2000; 96(2): 569 - 576. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. J. Kudryk, A. Bini, S. R. Kumar, and B. V. Zlokovic Monoclonal Antibody Designated T2G1 Reacts With Human Fibrin {beta}-Chain but Not With the Corresponding Chain From Mouse Fibrin Arterioscler Thromb Vasc Biol, July 1, 2000; 20(7): 1848 - 1849. [Full Text] [PDF]
|
|
|
|
|
|
C. D. Sigmund Viewpoint: Are Studies in Genetically Altered Mice Out of Control? Arterioscler Thromb Vasc Biol, June 1, 2000; 20(6): 1425 - 1429. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Ginsberg On Ischemic Brain Injury in Genetically Altered Mice Arterioscler Thromb Vasc Biol, November 1, 1999; 19(11): 2581 - 2583. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||