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Protective role of tuftsin fragment 1-3 in an animal model of intracerebral hemorrhage.

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Protective Role of Tuftsin Fragment 1-3 in
an Animal Model of
Intracerebral Hemorrhage
Jian Wang, MD, PhD, Andrew D. Rogove, MD, PhD, Anna E. Tsirka, MD, and Stella E. Tsirka, PhD
Intracerebral hemorrhage (ICH) causes morbidity and mortality and commonly follows the reperfusion after an ischemic
event. Tissue plasminogen activator (tPA), a fibrinolytic serine protease, is routinely given for the treatment of stroke.
However, tPA also can promote neuronal death, suggesting that caution should be exercised when using it. Furthermore,
tPA upon brain injury mediates microglial activation and modulates neuronal survival. To investigate the role of tPA and
microglia during brain hemorrhage, we induced experimentally ICH by intracerebral injection of collagenase. Seven days
after the introduction of ICH, it persisted in tPA-deficient (tPAⴚ/ⴚ) mice but is drastically reduced in size in wild-type
mice. Three weeks after ICH, there are still red blood cells in tPAⴚ/ⴚ but not in wild-type animals. Activated microglia
persist around the injury site. When microglial activation is inhibited by tuftsin fragment 1-3 macrophage/microglial
inhibitory factor (MIF), the stroke injury volume is significantly reduced, and the neurobehavioral deficits exhibited by
the mice are improved. Our results suggest that endogenous tPA assists in the clearance of intracerebral hemorrhage,
presumably by affecting microglial activation, and MIF could be a valuable neuroprotective agent for the treatment of
ICH.
Ann Neurol 2003;54:655– 664
Stroke is the second most common cause of death in
the world after heart disease and a leading cause of disability. Intracerebral hemorrhage (ICH) represents at
least 10% of all stroke deaths.1 The prognosis of patients after ICH is poor, and the pathogenesis of damage after ICH remains poorly understood. Evidence of
cell death due to apoptosis2,3 and inflammation has
been described recently.4 – 6
Tissue plasminogen activator (tPA) is a secreted
serine protease that converts inactive plasminogen to
the active serine protease plasmin. tPA and plasmin,
both members of the fibrinolytic system, initiate a potent proteolytic cascade that leads to dissolution of
blood clots.7 tPA remains the only Food and Drug Administration–approved treatment for acute stroke.8
However, the increased incidence of symptomatic ICH
has put constraints on tPA’s clinical use.9 Furthermore,
tPA and plasmin produced in the brain can promote
neuronal death after excitotoxin injection10 –13 or tran-
sient focal ischemia/reperfusion injury.14 tPA is expressed in the brain by neurons and by microglia, the
immunocompetent cells of the brain that have been associated when activated with neurotoxicity. tPA not
only is produced by microglia, but also mediates their
activation.11 When ICH occurs, the blood–brain barrier becomes disrupted. As a result, macrophages and
leukocytes infiltrate the brain parenchyma, and their
presence has been proposed to constitute a primary
mechanism of cell death. It is also possible that microglia contribute to the observed neuronal death, because
the actual disruption of blood–brain barrier can activate microglia.
To investigate the role of tPA and microglia in the
pathogenesis of the brain injury in ICH, we adapted
the mouse collagenase hemorrhage model.15 Extensive
activation of microglia was observed around the site of
ICH. When we used the tripeptide MIF (macrophage/
microglial inhibitory factor, tuftsin fragment 1-3,
From the Department of Pharmacological Sciences, University
Medical Center at Stony Brook, Stony Brook, NY.
Received Feb 3, 2003, and in revised form Jun 3. Accepted for
publication Jul 30, 2003.
Current address for Dr Rogove: Department of Neurology, New
York Presbyterian Hospital-Weill Cornell Medical Center, New
York, NY 10021.
Address correspondence to Dr Stella E. Tsirka, Department of Pharmacological Sciences, BST-7, Room 183, University Medical Center
at Stony Brook, Stony Brook, NY 11794-8651.
E-mail: stella@pharm.sunysb.edu
Current address for Dr Anna E. Tsirka: Department of Pediatrics,
Division of Pediatric Cardiology, University of Minnesota, Minneapolis, MN 55455.
© 2003 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
655
Thr-Lys-Pro) to inhibit the activation of microglia,14
functional improvement and decrease in degenerating
neurons were observed.
Materials and Methods
Animal Procedures
tPA-deficient (tPA⫺/⫺)16 and C57BL/6 (wild-type) mice
were maintained in the Department of Laboratory Animal
Research at Stony Brook with access to food and water ad
libitum. tPA⫺/⫺ mice have been backcrossed for 12 generations to the C57Bl/6 background. All experiments were
performed in accordance to the National Institutes of
Health guide for the care and use of laboratory animals as
well as the institutional guidelines established by the Institutional Animal Care and Use Committee (IACUC) committee at Stony Brook. All efforts were made to minimize
the use of animals and ensure minimal suffering of those
animals used.
saline (PBS). Injury volumes were digitally quantified, using
the NIH1.62 image software package, on 50␮m coronal sections using Luxol fast blue/cresyl violet staining.15 Hemorrhagic injury areas were summed from six to eight coronal
slices at different levels. Volumes in cubic millimeters were
calculated by multiplying the 0.5mm slice thickness by the
measured areas.
Histology
Hematoxylin staining was performed according to standard
protocols. Luxol fast blue/cresyl violet staining and the
Fluoro-Jade B (FJB) staining were performed according to
published protocols.15,17 Cells permeable to FJB were
marked for cell death. Degenerating neurons were counted in
three fields immediately adjacent to the hematoma using a
magnification of ⫻400 over a microscopic field of 0.1mm2
and expressed as cells per square millimeter; areas with large
blood vessels were avoided.
Immunofluorescence
Intracerebral Hemorrhage Model
The procedure for inducing ICH was adapted to mice from
an established rat protocol.15 In brief, mice weighing approximately 25 to 35gm were anesthetized by intraperitoneal injection of avertin (0.5mg/gm of body weight). To
induce hemorrhage, we injected mice with collagenase
(0.075U in 500nl saline) unilaterally into the caudate putamen, using the following stereotactic coordinates: 1.0mm
posterior and 3.0mm lateral of bregma, 4.0mm in depth.
Collagenase was delivered over 2 minutes, and we kept the
needle in place for an additional 5 minutes to prevent any
reflux.
Other mice were infused with 100␮l MIF (500␮M,
Sigma) in saline. MIF was delivered at a rate of 0.5␮l/hr
via a microosmotic pump (Durect, Cupertino, CA) placed
subcutaneously in the back of the animals. A brain infusion
cannula connected to the pump was positioned at the coordinates above to deliver the compound. Two days after
the onset of infusion, collagenase was injected unilaterally
into the caudate putamen of the mice; infusion of MIF
continued for another day. The mice were allowed to recover from surgery in a warm environment over a 3-hour
period. Mice were carefully observed and monitored for
several hours after recovery from anesthesia. MIF-treated
mice were killed 24 hours after the collagenase injection.
Non–MIF-treated mice were killed on days 1, 7, and 21
after injection.
Neurological Deficit
MIF-treated and control mice were scored blindly for neurological deficits using a 28-point neurological scoring system18 on day 1 after ICH. The tests included body symmetry, gait, climbing, circling behavior, front limb symmetry,
compulsory circling, and whisker response. Each point was
graded from 0 to 4. Maximum deficit score was 28.
Free-floating sections were washed in PBS for 20 minutes,
blocked in 5% normal serum, and incubated with 5-D-4 antibody (which recognizes activated microglia; 1:1,000; Seikagaku, East Falmouth, MA), followed by Alexa 488conjugated secondary antibody (1:1,000; Molecular Probes,
Eugene, OR). Control sections were processed as described
above, except that primary antibodies was omitted. Control
sections were devoid of specific staining (n ⱖ 3 for each
condition at each time point).
Microglial Cultures
Primary wild-type microglia were plated as described.19 Collagenase was added at different concentrations to these cells,
and their morphology was evaluated 24 hours later.
Spectrophotometric Assay for
Intracerebral Hemorrhage
The hemoglobin content of brains subjected to ICH was
quantified with Drabkin’s reagent. Brain hemispheres were
obtained from wild-type or tPA⫺/⫺ animals. The tissue (injected vs uninjected) was trimmed to contain only the caudate putamen region and was treated individually as follows.20 Distilled water (500␮l) was added to each sample,
followed by homogenization for 5 minutes, and centrifugation at 5,000g for 5 minutes. The supernatant, which contains the hemoglobin, was collected. Eighty microliters of
Drabkin’s reagent (Sigma, St. Louis, MO) was added to a
20␮l aliquot and allowed to stand for 15 minutes at room
temperature. The concentration of cyanmethemoglobin produced was measured at 550nm. A standard curve, reflecting
the amount of hemoglobin present, was generated by adding
to 100␮l lysate from untreated tissue 0.1, 0.5, 1.0, and 2.0␮l
of blood obtained from control mice by cardiac puncture
after anesthesia.
In Situ Matrix Metalloproteinases Activity
Hemorrhagic Injury Analysis
On day 1 after neurological scoring, mice were killed and
their brains were removed, fixed, and dehydrated in 4%
paraformaldehyde and 20% sucrose in phosphate-buffered
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To localize the gelatinolytic activity of matrix metalloproteinases (MMPs) in situ, we used fluorescein isothiocyanate–
labeled DQ gelatin as a substrate (EnzChek; Molecular
Probes). MMP activity yields fluorescent fluorescein isothio-
cyanate–conjugated gelatin. Fresh 10␮m cryostat sections
were incubated with reaction buffer (0.05M Tris-HCl,
0.15M NaCl, 5mM CaCl2, and 0.l2mM NaN3, pH 7.6)
containing 20␮g/ml DQ gelatin for 1 hour. MMP activity
was visualized by confocal microscopy and photographed.
MMP-expressing cells were counted in three fields immediately adjacent to the hematoma site by using a magnification
of ⫻400 over a microscopic field of 0.1mm2 and expressed
as cells/mm2; areas with large blood vessels were avoided. In
vitro collagenase activity was determined using the EnzChek
kit, as per manufacturer’s instructions.
Statistics
Statistical analysis was performed using SAS 8.0 software for
PC. The data were analyzed by one-way analysis of variance,
followed by multiple linear regression. Multiple regression
models included the interaction between tPA and MIF. Statistical significance was set at p value less than 0.05.
Results
The injection of collagenase is an established model of
ICH. Within 30 minutes, bleeding is observed at the
injection site and continues for 4 hours. No tissue necrosis is then evident. At 24 hours, two zones of injury
can be observed: a central zone of erythrocytes and necrotic tissue, surrounded by a peripheral zone that contains mixed viable and necrotic tissue. Forty-eight
hours after ICH, there is increased decay in the central
zone, and polymorphonuclear cells invade the periphery of the lesion.21 By 7 days, the central zone contains
necrotic ghost cells and pale erythrocytes, whereas the
peripheral zone contains a broad band of phagocytic
macrophages, and by 21 days the hemorrhagic area resolves to a cystic structure/glial scar bordered by reactive astrocytes.
Effects of Tissue Plasminogen Activator on
Intracerebral Hemorrhage Clearance
In tPA⫺/⫺ mice, an altered time course of hematoma
clearance was observed. Seven days after ICH, its
presence persisted in these mice. However, the hematoma was drastically reduced in size in wild-type
mice. By 21 days, red blood cells were still visible in
tPA⫺/⫺ mice, but not in wild-type mice (Fig 1).
These results indicate that wild-type and tPA⫺/⫺
mice show a different time course of clearance of the
hematoma, suggesting that endogenous tPA assists in
its clearance. Our results are in agreement with those
published recently in a study using a pig intracerebral
hemorrhage model.22
To ascertain that the difference in clearance is not
caused by a difference in the original collagenase injury, we measured the initial levels of hemoglobin in
the injected tissue in wild-type and tPA⫺/⫺ animals, as
Fig 1. Endogenous tissue plasminogen activator (tPA) assists in intracerebral hemorrhage (ICH) clearance. Coronal sections through
the site of collagenase delivery were collected at different time points after ICH and stained with hematoxylin. Wild-type and
tPA⫺/⫺ mice show a different time course of clearance of the hematoma. Note the presence of red blood cells, even at the later time
points in tPA⫺/⫺ mice. Magnification: left, ⫻100; right, ⫻400.
Wang et al: tPA, ICH, and Microglial Activation
657
an indicator of the bleeding introduced by the injection. No significant difference between wild-type and
tPA⫺/⫺ mice can be observed 18 to 24 hours after collagenase injection, indicating that the clearance delays
in tPA⫺/⫺ mice can be attributed only to the absence
of tPA (Fig 2).
Effect of Macrophage/Microglial Inhibitory Factor on
Microglial Activation after Intracerebral Hemorrhage
The persistence of the hematoma could be attributed
to the absence of the fibrinolytic properties of tPA/
plasmin, to limited phagocytic activity (because microglial activation is attenuated in tPA⫺/⫺ mice11), or to
both. We first evaluated the presence of activated microglia surrounding the injury site. Activated microglia
(5-D-4 –positive, round cells, Fig 3) were detected in
both wild-type and tPA⫺/⫺ mice 1 day after collagenase injection, but fewer activated microglia could be
seen in tPA⫺/⫺ mice. Seven days after ICH, activated
microglia persisted in tPA⫺/⫺, but their numbers were
drastically reduced in wild-type mice. By 21 days, the
Fig 2. The initial collagenase-induced intracerebral hemorrhage (ICH) is similar in wild-type and tPA⫺/⫺ mice. Total
hemoglobin levels were measured in lysates from the injected
caudate putamen of mice. A standard curve was made from
lysates from control (uninjected) animals. No significant difference in hemoglobin levels after induction of ICH was observed
between the two strains of mice (n ⫽ 4). tPA ⫽ tissue plasminogen activator.
numbers of activated microglial had returned to normal in wild-type mice but persisted in tPA⫺/⫺ mice.
Collagenase alone had no effect on microglial activation in culture (see Fig 3E).
Because there was a difference between wild-type
and tPA⫺/⫺ mice in microglial appearance and activation profile, we used an inhibitor of microglial activation before the injury. In an excitotoxic model, MIF
had prevented microglial activation.19 In the ICH paradigm, inhibition of microglial activation by MIF
(whose delivery started 2 days before the collagenase)
was evident 1 day after ICH both in wild-type and
tPA⫺/⫺ mice (see Fig 3). MIF had only a minimal inhibitory effect on collagenase, reducing its activity by
approximately 10% (Fig 4F).
Effect of Macrophage/Microglial Inhibitory Factor on
Stroke Volume and Neuronal Cell Death
Because MIF inhibited microglial activation, we evaluated its effect on injury volume and the ensuing
neuronal death. MIF treatment reduced injury
volume by 60 to 70% 1 day after ICH, from 9.5 ⫾
3.3 to 3.6 ⫾ 0.7mm3 in tPA⫺/⫺ mice ( p ⬍ 0.005),
and from 11.6 ⫾ 4.7 to 3.4 ⫾ 0.6mm3 in wild-type
mice ( p ⬍ 0.005), respectively (see Fig 4), indicating
that activated microglia contribute to injury during
ICH.
To examine whether neuronal death was evident at
the site of hemorrhage, we used the FJB histological
staining. MIF reduced the number of degenerating
neurons by nearly 50% (Fig 5), from 240 ⫾ 39/mm2
to 126 ⫾ 30/mm2 in tPA⫺/⫺ mice (n ⫽ 5; p ⬍
0.001) and from 272 ⫾ 61/mm2 to 152 ⫾ 31/mm2 in
wild-type mice (n ⫽ 5; p ⬍ 0.001).
Because there is no difference in degenerated neurons between tPA⫺/⫺ and wild-type mice, we suggest
that factors other than neuronal loss contribute to
morbidity/mortality after hemorrhage, that is, edema
leading to mass effect and herniation. Therefore, MIF
may be either affecting the edema volume or inhibiting the properties of infiltrating macrophages. Furthermore, persistent microglia could lead to additional cell death, thereby leading to additional
deficits.
Fig 3. Microglial activation after intracerebral hemorrhage. Microglial cells (5-D-4 –positive) are present near the hematoma in
wild-type and tPA⫺/⫺ mice. Microglial activation peaked in wild-type animals around day 7, but not in tPA⫺/⫺ mice, where significant microglial activation is evident at day 21. After infusion of macrophage/microglial inhibitory factor (MIF), microglial activation was efficiently inhibited at day 1 both in wild-type and tPA⫺/⫺ mice. Magnification: columns A, C, ⫻100; column B, D,
⫻400. Scale bars ⫽ 20␮m. (E) Addition of collagenase had no effect on microglial activation. LPS ⫽ lipopolysaccharide. tPA ⫽
tissue plasminogen activator.
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‹
Figure 3
Wang et al: tPA, ICH, and Microglial Activation
659
Fig 4. Macrophage/microglial inhibitory factor (MIF) reduced injury volume in wild-type and tPA⫺/⫺ mice. Coronal sections were
collected at day 1 after intracerebral hemorrhage (ICH) and stained for Luxol fast blue/cresyl violet. (A) Control (ctr) tPA⫺/⫺ mice;
(B) MIF-treated tPA⫺/⫺ mice; (C ) control wild-type mice; (D) MIF-treated wild-type mice. (E) MIF-treated wild-type (n ⫽ 5),
and tPA⫺/⫺ (n ⫽ 5) mice had smaller injury volumes than control-treated wild-type (n ⫽ 6) or tPA⫺/⫺ (n ⫽ 6) mice. Values
are means ⫾ SD. (F) Collagenase was incubated for different time periods with 0.5mM MIF or 1,10-phenanthroline (specific inhibitor), and its residual enzymatic activity was determined. tPA ⫽ tissue plasminogen activator; wt ⫽ wild type.
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Fig 5. Macrophage/microglial inhibitory factor
(MIF) reduced the number of neurons degenerating after intracerebral hemorrhage (ICH). (A)
Fluoro-Jade B (FJB) histological staining of neurons in coronal sections collected at different
time-points after the collagenase injection. Evenly
distributed, intensely labeled neurons and processes are observed in the peri-ICH region on
day 1 in wild-type and tPA⫺/⫺ mice. There is
persistence of degenerating neurons in tPA⫺/⫺
mice 7 days after ICH, but not in the wild-type
mice. Magnification: columns a, c: ⫻100; columns b, d: ⫻400. Scale bars ⫽ 20␮m. (B)
Quantification of the numbers of degenerating
neurons on day 1 in wild-type and tPA⫺/⫺ control and MIF-treated mice. MIF-treated mice
had fewer degenerating neurons than control
wild-type or tPA⫺/⫺ mice. n ⫽ 5 for each genotype. Values are means ⫾ SD.
Effect of Macrophage/Microglial Inhibitory Factor
on Intracerebral Hemorrhage–Induced
Neurobehavioral Deficits
Intracerebral hemorrhage usually is accompanied by
characteristic behavioral deficits. To determine
whether the ICH-induced pathological and molecular
events that showed improvement after MIF pretreatment were paralleled by neurobehavioral recovery, we
performed repeated assessments of the animals on day
1 after ICH. MIF significantly improved the neurobehavioral score of the animals compared with non–
MIF-treated animals; the score changed from 8.2 ⫾
0.8 to 5.4 ⫾ 1.1 in tPA⫺/⫺ mice ( p ⬍ 0.005) and
from 7.5 ⫾ 1.5 to 5.0 ⫾ 0.7 in wild-type mice ( p ⬍
0.005; Fig 6).
Matrix Metalloproteinase Activity after
Intracerebral Hemorrhage
The potential interaction between collagenase injury
and other proteolytic systems, especially other MMPs,
which could be activated by collagenase or plasmin,
Fig 6. Macrophage/microglial inhibitory factor (MIF) improved
neurological functional outcome on day 1 in both wild-type and
tPA⫺/⫺ mice, compared with control mice: from 8.2 to 5.4 in
tPA⫺/⫺, and from 7.5 to 5 in wild-type mice, respectively. Values are means ⫾ SD. tPA ⫽ tissue plasminogen activator.
Wang et al: tPA, ICH, and Microglial Activation
661
Fig 7. Matrix metalloproteinase (MMP) activation after intracerebral hemorrhage (ICH) on day 1 in control, wild-type, and
tPA⫺/⫺ mice. (A) MMP-expressing cells are present in the injury area in all animals. Magnification: columns a, c, ⫻100; columns
b, d: ⫻400. Scale bars ⫽ 20␮m. (B) Quantification of MMP-expressing cells on day 1 in wild-type and tPA⫺/⫺ mice. No significant difference was observed between the two genotypes of mice (n ⫽ 5). Values shown are means ⫾ SD.
was evaluated. MMP activity was determined in situ in
PBS-injected mice or in wild-type and tPA⫺/⫺ mice on
day 1 after ICH (Fig 7A). MMP activity was clearly
identifiable in the injury area of wild-type, tPA⫺/⫺,
and PBS-injected mice, indicating that the hemorrhagic injury can activate MMPs. The gelatinolytic activity appeared to be associated with neuronal cells. No
significant difference (271 ⫾ 41/mm2 vs 247 ⫾ 52/
mm2; p ⫽ 0.105) could be observed in MMP-
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expressing cells between wild-type (n ⫽ 5) and tPA⫺/⫺
mice (n ⫽ 5) on day 1 after ICH (see Fig 7B).
Discussion
Experimental and clinical studies have demonstrated
that tPA administration is beneficial when treatment is
initiated within 3 hours (intravenously) or up to 6
hours (intraarterial delivery) of the onset of ischemic
stroke.9,23 However, the presence of significant
amounts of tPA has resulted in increased incidence of
local ICH, which in turn has worsened the stroke outcome. The role of endogenous tPA in ICH is not clear.
Results obtained from a focal ischemia/reperfusion injury paradigm in the absence of induced thrombi in
tPA⫺/⫺ mice indicate that tPA contributes to neuronal
damage.10 The data here indicate that endogenous tPA
assists in ICH clearance but also affects microglial activation. Inhibition of microglial activation by MIF reduces the injury volume and degenerating neurons and
improves neurological functional outcome. Therefore,
when intraarterial tPA is injected to dissolve the clot, it
might be beneficial to coinject MIF, because it would
cross the permeable blood–brain barrier in stroke patients. In addition, it may be possible to wash the evacuated hemorrhage bed with MIF when ICH is treated
surgically.
Inflammation contributes to brain injury after
ICH.4 – 6 Our results indicate that, besides macrophage infiltration, microglial activation does occur
in and around hematomas. The presence of activated microglia also has been documented in another
model of ICH, in rats injected intracerebrally with
autologous blood.5 We show that there is a functional
consequence to the presence of activated microglia,
because MIF was able to reduce the cell death observed and improved the neurological functional outcome.
The inhibition of timely microglial activation results in a corresponding decrease in TNF-␣ and tPA
secretion.13,14,19 Consistent with the notion that MIF
inhibits microglial activation and macrophage migration, we show that MIF not only reduces the degenerating neurons and injury volume, but also improves
neurological functional outcome. Using multiple regression analysis, we correlated neuron density, lesion
volume, and neurological scores in wild-type and
tPA⫺/⫺ animals that had received control or MIF
treatment. As described in Results, MIF treatment
produced significant differences in all parameters examined ( p ⬍ 0.001, p ⬍ 0.005, and p ⬍ 0.005, respectively).
In conclusion, our study provides support for the
idea that activated microglia are important contributors
to brain injury during ICH. tPA, because of its thrombolytic role, is beneficial for hematoma clearance; however, its indirect role, microglial activation, should be
kept in check. In this respect, the recent report in experimental models of excitotoxicity using desmoteplase,
a vampire bat–derived tPA, provides an interesting
possibility of a thrombolytic enzyme devoid of microglial activation capabilities.24 Our results also suggest
that MIF could be a valuable neuroprotective agent for
the treatment of ICH. Further studies in other hemorrhagic stroke models are necessary to determine the optimal dose and administration of MIF in ICH.
This work was supported by grants from the National Institutes of
Health (ROINS042168, S.E.T.); a fellowship from the American
Heart Association (0225701T, J.W.), and by a National Institute of
Diabetes and Digestive and Kidney Diseases NRSA grant
(T32DK007521, A.D.R.).
We thank members of the Tsirka lab and Dr R. Thiex for critical
reading of the manuscript.
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