 |
 Previous Article | Next Article 
Volume 17, Number 23,
Issue of December 1, 1997
Delayed Reduction of Ischemic Brain Injury and Neurological
Deficits in Mice Lacking the Inducible Nitric Oxide Synthase Gene
Costantino Iadecola,
Fangyi Zhang,
Robyn Casey,
Masao Nagayama, and
M. Elizabeth Ross
Department of Neurology, University of Minnesota, Minneapolis,
Minnesota 55455
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Inducible nitric oxide synthase (iNOS), an enzyme that produces
toxic amounts of nitric oxide, is expressed in a number of brain
pathologies, including cerebral ischemia. We used mice with a null
mutation of the iNOS gene to study the role of iNOS in ischemic brain
damage. Focal cerebral ischemia was produced by occlusion of the middle
cerebral artery (MCA). In wild-type mice, iNOS mRNA expression in the
post-ischemic brain begun between 24 and 48 hr peaked at 96 hr and
subsided 7 d after MCA occlusion. iNOS mRNA induction was
associated with expression of iNOS protein and enzymatic activity. In
contrast, mice lacking the iNOS gene did not express iNOS message or
protein after MCA occlusion. The infarct and the motor deficits
produced by MCA occlusion were smaller in iNOS knockouts than in
wild-type mice (p < 0.05). Such reduction
in ischemic damage and neurological deficits was observed 96 hr after
ischemia but not at 24 hr, when iNOS is not yet expressed in wild-type
mice. The decreased susceptibility to cerebral ischemia in iNOS
knockouts could not be attributed to differences in the degree of
ischemia or vascular reactivity between wild-type and knockout mice.
These findings indicate that iNOS expression is one of the factors
contributing to the expansion of the brain damage that occurs in the
post-ischemic period. iNOS inhibition may provide a novel therapeutic
strategy targeted specifically at the secondary progression of ischemic
brain injury.
Key words:
cerebral ischemia;
knockout mice;
gene expression;
neuroprotection;
stroke;
reverse-transcription PCR
INTRODUCTION
Inducible nitric oxide synthase
(iNOS) is an enzyme that produces toxic levels of nitric oxide (NO)
(for review, see Gross and Wolin, 1995 ). NO produced by iNOS is
responsible for the toxicity of activated microglia-macrophages and
aggravates glutamate-mediated neuronal damage in vitro (Chao
et al., 1992; Dawson et al., 1994; Hewett et al., 1994). In brain, iNOS
is expressed in several pathological states, including tumors, trauma,
demyelination, AIDS dementia, Alzheimer's disease, and cerebral
ischemia (Endoh et al., 1994; Bagasra et al., 1995; Iadecola et al.,
1995b; Adamson et al., 1996; Clark et al., 1996; Hara et al., 1996;
Vodovotz et al., 1996). However, the role that NO produced by iNOS
plays in the mechanisms of these brain diseases has not been
defined.
In cerebral ischemia produced by occlusion of the rat middle cerebral
artery (MCA), iNOS expression begins 12 hr after induction of ischemia,
peaks at 48 hr, and subsides at 7 d (Iadecola et al., 1995b).
Investigations using the iNOS inhibitor aminoguanidine have suggested
that iNOS expression may contribute to ischemic brain injury (for
review, see Iadecola, 1997). Administration of aminoguanidine 24 hr
after MCA occlusion reduces the volume of the resulting infarct by
~30% (Iadecola et al., 1995a). These data raise the possibility that
NO produced by iNOS contributes to the late stages of ischemic brain
damage. However, this evidence is far from conclusive because
aminoguanidine also has neuroprotective effects unrelated to iNOS
inhibition. These include inhibition of advanced glycation endproducts
formation (Zimmerman et al., 1995) and inhibition of diamino oxidase,
an enzyme that produces toxic aldehydes from polyamines oxidation
(Brunton et al., 1994; Sessa and Perin, 1994). Therefore, the role of
iNOS expression in the mechanisms of cerebral ischemia remains to be
firmly established.
Mice with a null mutation of the iNOS gene have recently been developed
using homologous recombination (MacMicking et al., 1995; Wei et al.,
1995). These mice are relatively protected from hypotension and death
resulting from septic shock and from the deleterious effects of
carrageenin-induced local inflammation (MacMicking et al., 1995; Wei et
al., 1995). In the present study, therefore, we used iNOS null mice
(MacMicking et al., 1995) to investigate the role of iNOS expression in
ischemic brain damage. We found that the knockouts do not express iNOS
after focal cerebral ischemia, develop smaller infarcts, and have less
pronounced neurological deficits than wild-type controls. The reduction
in infarct volume and neurological deficits is observed at 96 hr but
not at 24 hr after induction of ischemia. The data provide
nonpharmacological evidence that NO produced by iNOS plays an important
role in the delayed progression of ischemic brain damage and provide
the rational basis for using inhibition of iNOS as a treatment for
stroke.
MATERIALS AND METHODS
Animals
All animal procedures were approved by the Animal Care Committee
of the University of Minnesota. C57BL/6 and SV129 mice were obtained
from Jackson Laboratories (Bar Harbor, ME). Breeding pairs of iNOS
knockouts were kindly provided by Drs. C. Nathan (Cornell University
Medical College) and J. Mudgett (Merck Research Laboratories)
(MacMicking et al., 1995). Mice were bred to obtain a colony of
homozygous iNOS knockouts. After each experiment, knockouts were
genotyped to verify the lack of the iNOS gene and the presence of the
targeting vector.
Induction of focal cerebral ischemia
Focal cerebral ischemia was produced by occlusion of the MCA
(Zhang and Iadecola, 1992). Mice were anesthetized with 2% halothane in 100% oxygen. Body temperature was maintained at 37 ± 0.5°C by a thermostatically controlled infrared lamp. A 2 mm hole was drilled
in the inferior portion of the temporal bone to expose the left MCA.
The MCA was elevated and cauterized distal to the origin of the
lenticulostriate branches (Zhang and Iadecola, 1992). Wounds were
sutured, and mice were allowed to recover and then returned to their
cages. Rectal temperature was controlled until mice regained full
consciousness. Mice were killed at different time points after MCA
occlusion for determination of mRNA, for the iNOS assay, for
immunocytochemistry, or measurement of infarct size (see below).
Determination of mRNA by reverse transcription (RT)-PCR
iNOS or COX-2 mRNA was detected in the ischemic brain using
RT-PCR, as described previously (Iadecola et al., 1996; Ross and Iadecola, 1996; Nogawa et al., 1997). Briefly, a 2-mm-thick coronal brain slice was cut at the level of the optic chiasm, and the infarcted
cortex was dissected using the corpus callosum as a ventral landmark.
The homotopic region of the contralateral cortex was also sampled.
Total RNA was extracted, and the integrity of the RNA was determined on
denaturing formaldehyde gels. First-strand cDNA synthesis was then
performed using 0.25, 0.5, and 1.0 µg of total RNA, oligo dT primer
(BRL, Bethesda, MD) and Moloney Murine leukemia virus reverse
transcriptase (New England BioLabs, Beverly, MA). Aliquots (5 µl
each) from the RT reaction were then used for PCR amplification with
primer pairs for the mRNA of interest and the ubiquitously expressed
control sequence porphobilinogen deaminase (PBD). The iNOS
primer sequences used were Forward, 5 -ACAACGTGAAGAAAACCCCTTGTG-3;
Reverse, 5-ACAGT TCCGAGCGTCAAAGACC-3. The COX-2 primers were
Forward, 5-CCATGTCAAAACCGTGGTGAATG-3; Reverse,
5-ATGGG AGTTGGGCAGTCATCAG-3. The "hot start" method was used
(Stratagene, La Jolla, CA) with the following cycle parameters: 94°C,
15 sec; 65°C, 30 sec; 73°C, 25 sec, × 5 cycles; then 94°C, 15 sec; 60°C, 30 sec; 73°C, 25 sec, × 35 cycles. Reaction products were then separated on an 8% acrylamide gel, ethidium-stained, and
photographed. The PCR products were sequenced to confirm their identity. Each set of PCR reactions included control samples run without RNA/RT. Relative optical density (OD) of the bands was measured
using an image analysis system (M4, Imaging Research Inc.). All
measurements were normalized to the relative OD of the PBD band
(Iadecola et al., 1995b; Nogawa et al., 1997).
iNOS enzymatic activity and immunocytochemistry
iNOS catalytic activity of the brain samples was determined by
the citrulline assay of Bredt and Snyder modified for detection of
calcium-independent activity as described previously (Ross and
Iadecola, 1996). Immunocytochemical procedures were identical to those
described previously (Iadecola et al., 1996; Nogawa et al., 1997).
Sections (7 µm) from formalin-fixed, paraffin-embedded brains were
incubated overnight (4°C) with an iNOS polyclonal antibody (Upstate
Biotechnology, Lake Placid, NY) (dilution 1:200), washed, and incubated
with the secondary antibody (Vector, Laboratories, Burlingame, CA) for
30 min. The immunocomplex was visualized using the ABC complex
(Vectastain Elite Kit, Vector). Alternate sections were processed for
immunocytochemistry for glial fibrillary acidic protein (GFAP)
(Boehringer Mannheim, Indianapolis, IN) or myeloperoxidase (Dako,
Carpinteria, CA).
Determination of infarct volume
Mice were killed 24 or 96 hr after MCA occlusion. Brains were
removed and frozen in cooled isopentane ( 30°C). Coronal forebrain sections (thickness 30 µm) were serially cut in a cryostat, collected at 150 µm intervals, and stained with thionin for determination of
infarct volume by an image analyzer (MCID, Imaging Research) (Zhang and
Iadecola, 1992). To factor out the contribution of ischemic edema to
the total volume of the lesion, infarct volume in cerebral cortex was
corrected for swelling as described previously (Zhang and Iadecola,
1994; Iadecola et al., 1995a). The correction method is based on the
determination of ischemic swelling by comparing the volume of ischemic
and nonischemic hemispheres (Lin et al., 1993).
Determination of neurological deficits
Neurological deficits were assessed by a neurological scoring
system widely used in mice (Huang et al., 1994; Yang et al., 1994).
Because distal MCA occlusion produced no neurological deficits measurable by this scale, in these experiments the MCA was occluded at
a more proximal site, a procedure that results in a larger infarct area
and measurable neurological deficits. The examiner was not aware of the
identity of the mice. The neurological scores were as follows: 0, normal motor function; 1, flexion of torso and contralateral forelimb
when mouse was lifted by the tail; 2, circling to the contralateral
side when mouse was held by the tail on a flat surface, but normal
posture at rest; 3, leaning to the contralateral side at rest; 4, no
spontaneous motor activity (Huang et al., 1994; Yang et al., 1994).
Mice were evaluated before MCA occlusion and at 24 hr intervals up to
96 hr after MCA occlusion.
Determination of cerebral blood flow (CBF)
Techniques used for studying the cerebral circulation in mice
were similar to those described previously (Dalkara et al., 1995; Sobey
and Faraci, 1997). Mice were anesthetized with halothane (maintenance
1%), and the femoral artery and trachea were cannulated. Mice were
artificially ventilated with an oxygen-nitrogen mixture by a
mechanical ventilator (SAR-830, CWI Inc., Ardmore, PA). The inspiration
time was set at 0.1 sec, the respiratory rate at 120/min, and the
inspiratory flow at ~250 ml/min. The oxygen concentration in the
mixture was adjusted to obtain an arterial PO2
of 150-170 mmHg. End-tidal CO2 was monitored continuously
using a CO2 analyzer (Capstar-100, CWI Inc.). The sample
flow rate of the CO2 analyzer was set at 10 cc/min.
Throughout the experiment, end-tidal CO2 was maintained at
2.6-2.7%, which corresponds to a PCO2 of
33-35 mmHg, as determined in preliminary studies in which end-tidal CO2 was compared with arterial PCO2
measured by a blood gas analyzer.
MCA occlusion. For monitoring of the changes in CBF produced
by MCA occlusion, two laser-Doppler flow probes (Vasamedic, St. Paul,
MN) were inserted through burr holes placed in the center (3.5 mm
lateral to the midline and 1 mm caudal to bregma) and the periphery
(1.5 mm lateral to the midline and 1.7 mm rostral to lambda) of the
ischemic territory (Chan et al., 1993). The location of the probe was
selected in preliminary experiments to correspond to the region of
brain that is spared from infarction in the iNOS knockouts. After
placement of the probes, the MCA was occluded, and CBF was monitored
for 90 min. CBF data are expressed as percentage of the preocclusion
value.
Cerebrovascular reactivity to hypercapnia. Techniques for
testing cerebral vascular reactivity to hypercapnia in mice were similar to those described previously in rats (Iadecola, 1992; Iadecola
and Zhang, 1996). Mice were anesthetized and instrumented as described
above. CBF was monitored continuously in the frontoparietal cortex with
a laser-Doppler probe. After stabilization of arterial pressure and
blood gases, CO2 was introduced into the circuit of the
ventilator, and the increase in CBF produced by hypercapnia was
monitored. CO2 administration was discontinued after the
CBF increase reached a plateau (usually 2-3 min).
Data analysis
Data in text and figures are expressed as mean ± SE.
Multiple comparisons were statistically evaluated by the ANOVA and
Tukey's test. Two-group comparisons were analyzed by the two-tailed
t test for independent samples. Neurological scores were
evaluated by nonparametric statistical procedures. Two group
comparisons were analyzed by the Mann-Whitney U analysis
(Huang et al., 1994; Yang et al., 1994), and multiple contrasts were
analyzed by the Kruskal-Wallis ANOVA followed by the Tukey-Kramer
test (Systat, Evanston, IL). For all procedures, probability values of
<0.05 were considered statistically significant.
RESULTS
iNOS expression in wild-type and null mice
In wild-type mice, occlusion of the MCA induced expression of iNOS
message (n = 4/time point) and enzymatic activity
(n = 4/time point) (Fig.
1). mRNA expression began between 24 and
48 hr, peaked at 96 hr, and subsided 7 d after MCA occlusion (Fig. 1A). iNOS immunoreactivity was observed in mono- and
polynuclear inflammatory cells invading the infarct border (Fig.
2B). In contrast, MCA
occlusion did not induce expression of message or protein in iNOS
knockouts (Fig. 2A,B).
Fig. 1.
Expression of iNOS message and enzymatic activity
after cerebral ischemia in B6 mice. A, Expression of
iNOS message after focal cerebral ischemia. iNOS mRNA was determined by
RT-PCR. A strong iNOS signal is observed at 48 and 96 hr after ischemia (top panel). The bottom panel
shows the time course of the ratio of the optical density of the iNOS
band by the density of the band corresponding to porphobilinogen
deaminase (PBD), a housekeeping gene used for
normalization. Significant elevation is observed at 48 and 96 hr after
ischemia (p < 0.05; ANOVA and Tukey's
test). No expression is observed at 24 hr or 7 d
(p > 0.05). B, iNOS enzymatic activity after cerebral ischemia. iNOS activity was measured
using the citrulline conversion assay of Bredt and Snyder modified for
detection of calcium-independent arginine-to-citrulline conversion,
i.e., iNOS activity (Ross and Iadecola, 1996). Virtually no iNOS
activity was observed in sham-operated mice. Substantial iNOS activity
developed in the ischemic region 96 hr after MCA occlusion. The 96 hr
time point was chosen because at this time iNOS message is maximal.
These findings suggest that cerebral ischemia leads to expression of
iNOS message and enzymatic activity in the injured brain.
[View Larger Version of this Image (34K GIF file)]
Fig. 2.
A, iNOS message detected by RT-PCR
in wild-type mice (C57/B6) and iNOS knockouts at 48 and 96 hr after
ischemia. No iNOS signal is detected in the ischemic brain at either
time points. B, Immunoreactivity for
iNOS, glial fibrillary acidic protein
(GFAP), or myeloperoxidase (MPO) in iNOS
null mice and controls (C57/B6) 96 hr after MCA occlusion. Cerebral ischemia is followed by expression of iNOS immunoreactivity in C57/B6 mice but not in iNOS knockouts (top panels). Immunoreactivity for the astrocytic marker GFAP
(middle panels) is comparable between iNOS null mice and
wild-type controls. The asterisk indicates the infarcted
area. GFAP expression occurs at the border of the lesion. To detect
infiltrating inflammatory cells in the post-ischemic brain,
immunocytochemistry for MPO, an enzyme expressed in polymorphonuclear
cells infiltrating the ischemic brain, was performed. Infiltrating
myeloperoxidase-positive cells are observed both in iNOS null mice and
in controls (bottom panels). Scale bar (shown in
top right panel in B): top
and bottom panels, 50 µm; middle panel,
250 µm.
[View Larger Version of this Image (73K GIF file)]
To determine whether other inflammation-related genes were upregulated
in the iNOS null mice, the mRNA expression of cyclooxygenase-2 (COX-2)
was studied by RT-PCR. COX-2 is a prostaglandin-synthesizing enzyme
that is induced in inflammatory states, including post-ischemic inflammation (Nogawa et al., 1997). Forty-eight hours after MCA occlusion, COX-2 mRNA was upregulated equally in iNOS null mice (0.25 ± 0.05 relative OD; n = 4) and in wild-type
controls (0.23 ± 0.06; n = 4; p > 0.05; t test). The infiltration of neutrophils, as
revealed by myeloperoxidase immunocytochemistry, and the expression of
the astroglial marker GFAP were comparable in iNOS null mice and
wild-type controls (Fig. 2B). These data indicate
that iNOS expression does not occur in the post-ischemic brain of iNOS
null mice, whereas COX-2 expression and the cellular reaction to
cerebral ischemia are not affected.
Infarct volume in wild-type and iNOS null mice
We then studied the outcome of cerebral ischemia in iNOS null
mice. The MCA was occluded, and the volume of the infarct was measured
96 hr later. Infarct volume was 28% smaller in iNOS knockouts than in
wild-type controls (C57/B6 strain) (Figs.
3, 4). The
genetic background of iNOS knockouts is mixed, including both the
C57/B6 and SV129 strains (MacMicking et al., 1995). To examine the
possibility that the reduction in ischemic damage was related to a
reduced susceptibility to ischemic injury of the SV129 strain, we
compared stroke size in iNOS null mice and SV129 mice. Cortical infarct volumes (corrected for swelling) in iNOS null mice (18 ± 1 mm3; n = 6) were also smaller than
those of SV129 mice (23 ± 0.6 mm3;
n = 6; p < 0.05).
Fig. 3.
A, Infarct volume in wild-type
controls (C57/B6) and iNOS knockouts killed 96 hr after occlusion of
the MCA. Infarct size is smaller in iNOS knockouts
(p < 0.01; t test). The
difference persists after correction for ischemic swelling
[Cortex (E.C.)]. B, Distribution of the
infarcted areas at different rostrocaudal levels in wild-type controls
(C57/B6) and iNOS knockouts. The area of infarction is smaller in the
iNOS knockouts at all rostrocaudal levels. Values for iNOS null mice
and controls were compared at each rostrocaudal level by the unpaired
t test.
[View Larger Version of this Image (21K GIF file)]
Fig. 4.
Representative brain sections illustrating the
distribution of the ischemic damage in wild-type mice (C57/B6) and iNOS
knockouts 96 hr after MCA occlusion. The area of infarction involves
the cerebral cortex almost exclusively. Note that the infarct is
smaller in the knockouts and that the area spared from infarction
involves the peripheral regions of the infarct. This peripheral region represents the so-called ischemic penumbra.
[View Larger Version of this Image (26K GIF file)]
The observation that iNOS expression occurs >24 hr after
cerebral ischemia (Fig. 1) suggests that iNOS may play a role in the
delayed evolution of the damage. To provide evidence in support of this
hypothesis, infarct volume was determined in iNOS null mice killed 24 hr after MCA occlusion. At this time, iNOS is not yet expressed in the
post-ischemic brain of wild-type controls (Fig. 1A).
At 24 hr after MCA occlusion, stroke volume in iNOS null mice was not
reduced compared with wild-type controls (Fig. 5A). Therefore, the reduction
in infarct size in iNOS knockouts is observed 96 hr but not 24 hr after
MCA occlusion.
Fig. 5.
A, Infarct volume in wild-type
controls (C57/B6) and iNOS knockouts killed 24 hr after occlusion of
the MCA. Data were corrected for ischemic swelling [Cortex
(E.C.)]. Infarct volume is not different between wild-type
mice and iNOS knockouts (p > 0.05;
t test). B, Neurological deficits in
wild-type mice (C57/B6) and iNOS knockouts. Mice were examined before
induction of ischemia and at 24 hr intervals until the time they were
killed 96 hr after MCA occlusion. Higher neurological scores indicate
more severe deficits. Neurological scores were not different 24 hr
after MCA occlusion (p > 0.05; Mann-Whitney U test). However, although the scores
remained stable in wild-type mice during the following 96 hr
(p > 0.05; Kruskal-Wallis ANOVA), they
improved markedly in the iNOS knockouts (p < 0.05 from wild-type mice at 96 hr and p < 0.05 from 24 hr).
[View Larger Version of this Image (20K GIF file)]
Motor deficits in wild-type and iNOS null mice
To determine whether the reduction in infarct size corresponds to
a functional improvement, mice were evaluated using a neurological scale for rodents (Huang et al., 1994; Yang et al., 1994). Twenty-four hours after ischemia, neurological scores did not differ between wild-type controls (C57/B6) and knockouts (Fig. 5B)
(p > 0.05). Although the deficits remained
stable between 24 and 96 hr in controls (p > 0.05; Kruskal-Wallis ANOVA), in iNOS knockouts the deficits improved
steadily. At 96 hr after MCA occlusion, the deficits were significantly
smaller than those of wild-type mice (p < 0.05;
Mann-Whitney U test). Therefore, the iNOS knockouts have a
better functional outcome after MCA occlusion.
CBF in wild-type and iNOS null mice
In these experiments, the effect of MCA occlusion on CBF and the
CBF reactivity to hypercapnia were studied in wild-type and iNOS null
mice. MCA occlusion resulted in a comparable flow reduction in
wild-type mice (C57/B6) and iNOS knockouts both in the center and at
the periphery of the ischemic territory (Fig.
6). Similarly, in intact mice, the
increase in CBF produced by hypercapnia (PCO2 = 50-60 mmHg) did not differ between wild-type controls and iNOS null
mice (wild type, +74 ± 3%; null mice, +71 ± 3% of
baseline; p > 0.05; n = 6/group).
These data indicate that the degree of ischemia induced by MCA
occlusion and the vascular reactivity to hypercapnia are comparable
between wild-type mice and iNOS knockouts.
Fig. 6.
A, Effect of MCA occlusion on CBF
in iNOS knockouts and wild-type controls (C57/B6). The reduction in CBF
produced by MCA occlusion is similar in the two groups both in the
center of the ischemic territory (Core) and at the
periphery (Penumbra). B, Arterial
pressure of the mice before and after MCA occlusion. Arterial pressure
did not differ between controls and iNOS knockouts (p > 0.05).
[View Larger Version of this Image (19K GIF file)]
DISCUSSION
We have demonstrated that knockout mice lacking the iNOS gene do
not express iNOS message or protein after MCA occlusion and have
smaller infarcts and reduced motor deficits in comparison to wild-type
controls. The effect is not observed 24 hr after ischemia but becomes
apparent at 96 hr. These data provide nonpharmacological evidence that
iNOS expression contributes to the development of the tissue damage and
neurological deficits resulting from cerebral ischemia.
The reduction in infarct size cannot be attributed to
hemodynamic-vascular factors, because the CBF decrease produced by MCA occlusion and the reactivity of the cerebral circulation to hypercapnia did not differ between wild-type and knockouts. Similarly, the protection in the knockouts is not caused by a reduced inflammatory reaction, because the expression of another inflammation-related gene,
COX-2, was not reduced in the knockouts. In addition, the degree of
cellular infiltration and glial activation was comparable in wild-type
and knockout mice. Strain differences in susceptibility to ischemic
injury are also unlikely to play a role, because the infarct in the
knockouts was smaller than that of both C57/B6 and SV129 mice.
Therefore, the reduction in ischemic damage in the null mice is likely
to result from the absence of iNOS expression and not from other
factors. It must be pointed out, however, that comparisons with both
parent strains individually do not completely rule out factors related
to the mixed genetic background of the null mice (Gerlai, 1996). In
addition, compensatory changes in the null mice secondary to the lack
of the iNOS gene cannot be excluded.
The expression of iNOS begins >24 hr after induction of ischemia and
peaks at 96 hr. Furthermore, the reduction in infarct size and the
improvement in neurological deficits is observed at 96 hr but not at 24 hr after MCA occlusion. These data indicate that iNOS does not
participate in the initiation of ischemic brain damage. Rather, the
evidence suggests that iNOS is involved in the mechanisms of the
delayed evolution of the damage that occurs after focal cerebral
ischemia. Most studies to date have focused on pathogenic factors
acting in the early stages of ischemic brain damage. Thus, glutamate
excitotoxicity, calcium overload, and free radical damage have been
extensively investigated as factors initiating ischemic brain death
(Choi, 1990; Siesjo, 1994; Chan, 1996). Recent evidence, however,
indicates that the ischemic damage continues to progress well after the
onset of ischemia. For example, magnetic resonance imaging (MRI) and
positron emission tomography (PET) studies have demonstrated that the
area of irretrievable damage increases in size for days after the onset
of ischemia (Heiss et al., 1992; Furlan et al., 1996; Marchal et al.,
1996; Baird et al., 1997). This evidence has challenged the concept of
a rigid therapeutic window of 4-6 hr and has raised the possibility that treatments can be instituted many hours after the onset of ischemia (Baron et al., 1995). The findings of the present study identify iNOS expression as one of the factors contributing to the
delayed evolution of ischemic brain damage. In particular, they suggest
that large amounts of NO produced by iNOS in inflammatory cells
infiltrating ischemic tissue contribute to the expansion of the infarct
that occurs in the post-ischemic period.
In apparent contrast with the hypothesis that ischemic brain damage
evolves during the post-ischemic period is the observation that the
infarct volume of wild-type mice, as estimated by the area of pallor in
thionin-stained sections, does not increase between 24 and 96 hr after
MCA occlusion (compare Figs. 3 and 5). Similarly, the neurological
deficits do not worsen during this period. These findings, however, are
not surprising, because the pale area at 24 hr represents brain tissue
that is functionally compromised but not entirely necrotic (Garcia et
al., 1995). Although necrosis develops rapidly in the center of the
ischemic territory, it progresses at a much slower pace at the
periphery of the infarct (Dereski et al., 1993; Garcia et al., 1993).
MRI and PET studies have demonstrated that the volume of permanently
damaged brain grows over several days after ischemia (Heiss et al.,
1992; Baird et al., 1997). These imaging data, therefore, support the
histological evidence of a slow progression of irreversible tissue
damage at the periphery of the ischemic territory. The lack of
progression of the neurological deficits between 24 and 96 hr can be
attributed to the fact that the deficits are already maximal at 24 after MCA occlusion because of ischemia-induced brain dysfunction
(Astrup et al., 1977). Although in wild-type mice the volume of pallor remains stable between 24 and 96 hr, in the knockouts, the volume at 96 hr is smaller than at 24 hr. The reduction in lesion size in the
post-ischemic period indicates that the growth of the infarct is less
pronounced in the knockouts. Importantly, the reduction in infarct
volume is accompanied by an improvement in neurological deficits. The
correspondence between reduction in infarct volume and neurological
deficits in the knockouts indicates that the brain tissue spared from
infarction is indeed functional.
Previous studies have indicated that NO is also involved in the acute
stages of ischemic brain damage (Dawson et al., 1991; Nowicki et al.,
1991; for review, see Iadecola, 1997). Shortly after induction of
ischemia, NO is thought to be produced in response to activation of
glutamate receptors, which in turn leads to sustained activation of
neuronal NOS (Malinski et al., 1993; Lin et al., 1996). Accordingly,
early treatment with inhibitors of neuronal NOS reduces ischemic
damage, whereas knockout mice lacking the neuronal NOS gene have
smaller infarcts (Huang et al., 1994; Yoshida et al., 1994; Nagafuji et
al., 1995; Zhang et al., 1996). The present study suggests that NO
plays a role also in the late stages of cerebral ischemia. Therefore,
NO contributes to both the initiation and the progression of ischemic
injury.
The mechanisms by which NO produced by iNOS contributes to ischemic
brain damage are unknown. However, it is well established that large
amounts of NO are cytotoxic, an effect exerted through multiple
mechanisms. These include peroxynitrite-mediated oxidative damage, DNA
damage, and energy failure resulting from inhibition of critical
energy-producing enzymes and from poly(ADP-ribose)synthase activation
(Beckman et al., 1990; Nguyen et al., 1992; Zhang et al., 1994; Gross
et al., 1996). It is therefore likely that NO produced by iNOS in the
late post-ischemic period affects the survival of potentially viable
neurons at the infarct border. This suggestion is strengthened by the
observation that iNOS expression worsens the neurotoxicity produced by
activation of glutamate receptors or oxygen-glucose deprivation in
neuronal cultures (Dawson et al., 1994; Hewett et al., 1994, 1996).
The findings of the present study have important implications for the
pathophysiology and treatment of ischemic stroke. First, they provide
evidence that ischemic brain damage evolves over a period of days and
that retrievable brain tissue is present >12 hr after the onset of
ischemia. This conclusion is also supported by recent imaging studies
in humans demonstrating that ~30% of ischemic brain tissue remains
viable for >12 hr after the onset of ischemia (Furlan et al., 1996;
Marchal et al., 1996). Second, the study identifies iNOS as one of the
factors contributing to the post-ischemic evolution of the stroke and
provides the rationale for therapeutic interventions based on iNOS
inhibition. Treatments based on iNOS inhibition could be implemented in
the late stages of cerebral ischemia (12-24 hr after stroke), at a
time when current therapeutic approaches are no longer efficacious
(Koroshetz and Moskowitz, 1996). iNOS inhibitors could complement
interventions, such as thrombolysis or glutamate receptor inhibition,
directed at the early stages of cerebral ischemia. Targeting both the
early and late pathogenic components of cerebral ischemia is likely to
be a successful strategy for the treatment of ischemic stroke.
FOOTNOTES
Received Aug. 4, 1997; revised Sept. 18, 1997; accepted Sept. 18, 1997.
This work was supported by grants from the American Heart Association
and National Institutes of Health Grants NS34179 and NS35806. C.I. is
an Established Investigator of the American Heart Association. We thank
Ms. Colleen Forster for the histological analysis and Ms. Karen MacEwan
for editorial assistance.
Correspondence should be addressed to Dr. C. Iadecola, Department of
Neurology, University of Minnesota, Box 295 UMHC, 420 Delaware Street
SE, Minneapolis, MN 55455.
REFERENCES
-
Adamson DC,
Wildemann B,
Sasaki M,
Glass JD,
McArthur JC,
Christov VI,
Dawson TM,
Dawson VL
(1996)
Immunologic NO synthase: elevation in severe AIDS dementia and induction by HIV-1 gp41.
Science
274:1917-1921[Abstract/Free Full Text].
-
Astrup J,
Symon L,
Branston NM,
Lassen NA
(1977)
Cortical evoked potential and extracellular K+ and H+ at critical levels of brain ischemia.
Stroke
8:51-57[Abstract/Free Full Text].
-
Bagasra O,
Michaels FH,
Zheng Y-M,
Bobroski LE,
Spitsin SV,
Fu F,
Tawadros R,
Koprowski H
(1995)
Activation of the inducible form of nitric oxide synthase in the brains of patients with multiple sclerosis.
Proc Natl Acad Sci USA
92:12041-12045[Abstract/Free Full Text].
-
Baird AE,
Benfield A,
Schlaug G,
Siewert B,
Lovblad KO,
Edelman RR,
Warach S
(1997)
Enlargement of human cerebral ischemic lesion volumes measured by diffusion-weighted magnetic resonance imaging.
Ann Neurol
41:581-589[Web of Science][Medline].
-
Baron J-C,
von Kummer R,
del Zoppo GJ
(1995)
Treatment of acute ischemic stroke. Challenging the concept of a rigid and universal time window.
Stroke
26:2219-2221[Free Full Text].
-
Beckman JS,
Beckman TW,
Chen J,
Marshall PA,
Freeman BA
(1990)
Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide.
Proc Natl Acad Sci USA
87:1620-1624[Abstract/Free Full Text].
-
Brunton VG,
Grant MH,
Wallace HM
(1994)
Spermine toxicity in BHK-21/C13 cells in the presence of bovine serum: effect of aminoguanidine.
Toxicol In Vitro
8:337-341.
-
Chan PH
(1996)
Role of oxidants in ischemic brain damage.
Stroke
27:1124-1129[Abstract/Free Full Text].
-
Chan PH,
Kamii H,
Yang G,
Gafni J,
Epstein CJ,
Carlson E,
Reola L
(1993)
Brain infarction is not reduced in SOD-1 transgenic mice after a permanent focal cerebral ischemia.
NeuroReport
5:293-296[Web of Science][Medline].
-
Chao CC,
Hu S,
Molitor TW,
Shaskan EG,
Peterson PK
(1992)
Activated microglia mediate neuronal cell injury via a nitric oxide mechanism.
J Immunol
149:2736-2741[Abstract].
-
Choi DW
(1990)
Cerebral hypoxia: some new approaches and unanswered questions.
J Neurosci
10:2493-2501[Web of Science][Medline].
-
Clark RSB,
Kochanek PM,
Schwartz MA,
Schiding JK,
Turner DS,
Chen M,
Carlos TM,
Watkins SC
(1996)
Inducible nitric oxide expression in cerebrovascular smooth muscles and neutrophils after traumatic brain injury.
Pediatr Res
39:784-790[Web of Science][Medline].
-
Dalkara T,
Irikura K,
Huang Z,
Panahian N,
Moskowitz MA
(1995)
Cerebrovascular responses under controlled and monitored physiological conditions in the anesthetized mice.
J Cereb Blood Flow Metab
15:631-638[Web of Science][Medline].
-
Dawson VL,
Dawson TM,
London ED,
Bredt DS,
Snyder SH
(1991)
Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures.
Proc Natl Acad Sci USA
88:6368-6371[Abstract/Free Full Text].
-
Dawson VL,
Brahmbhatt,
Mong JA,
Dawson TM
(1994)
Expression of inducible nitric oxide synthase causes delayed neurotoxicity in primary mixed neuronal-glial cortical cultures.
Neuropharmacology
33:1425-1430[Web of Science][Medline].
-
Dereski MO,
Chopp M,
Knight RA,
Rodolosi LC,
Garcia JH
(1993)
The heterogeneous temporal evolution of focal ischemic neuronal damage in the rat.
Acta Neuropathol
85:327-333[Medline].
-
Endoh M,
Maiese K,
Wagner J
(1994)
Expression of the inducible form of nitric oxide synthase by reactive astrocytes after transient global ischemia.
Brain Res
651:92-100[Web of Science][Medline].
-
Furlan M,
Marchal G,
Viader F,
Derlon J-M,
Baron J-C
(1996)
Spontaneous neurological recovery after stroke and the fate of the ischemic penumbra.
Ann Neurol
40:216-226[Web of Science][Medline].
-
Garcia JH,
Yoshida Y,
Chen H,
Li Y,
Zhang ZG,
Lian J,
Chen S,
Chopp M
(1993)
Progression from ischemic injury to infarct following middle cerebral artery occlusion in the rat.
Am J Pathol
142:623-635[Abstract].
-
Garcia JH,
Liu KF,
Ho KL
(1995)
Neuronal necrosis after middle cerebral artery occlusion in Wistar rats progresses at different time intervals in the caudoputamen and the cortex.
Stroke
26:636-642[Abstract/Free Full Text].
-
Gerlai R
(1996)
Gene-targeting studies of mammalian behavior: is it the mutation or the background genotype?
Trends Neurosci
19:177-181[Web of Science][Medline].
-
Gross SS,
Wolin MS
(1995)
Nitric oxide: pathophysiological mechanisms.
Annu Rev Physiol
57:737-769[Web of Science][Medline].
-
Gross WL,
Bak MI,
Ingwall JS,
Arstall MA,
Smith TW,
Balligand J-L,
Kelly RA
(1996)
Nitric oxide inhibits creatine kinase and regulates rat heart contractile reserves.
Proc Natl Acad Sci USA
93:5604-5609[Abstract/Free Full Text].
-
Hara E,
Takahashi K,
Tominaga T,
Kumabe T,
Kayama T,
Suzuki H,
Fujita H,
Yoshimoto T,
Shirato K,
Shibahara S
(1996)
Expression of heme oxygenase and inducible nitric oxide synthase mRNA in human brain tumors.
Biochem Biophys Res Commun
224:153-158[Web of Science][Medline].
-
Heiss W-D,
Huber M,
Fink GR,
Herloz K,
Pietrzyk U,
Wagner R,
Weinhard K
(1992)
Progressive derangement of periinfarct viable tissue in ischemic stroke.
J Cereb Blood Flow Metab
12:193-203[Web of Science][Medline].
-
Hewett SJ,
Csernansky CA,
Choi DW
(1994)
Selective potentiation of NMDA-induced neuronal injury following induction of astrocytic iNOS.
Neuron
13:487-494[Web of Science][Medline].
-
Hewett SJ,
Muir JK,
Lobner D,
Symons A,
Choi DW
(1996)
Potentiation of oxygen-glucose deprivation-induced neuronal death after induction of iNOS.
Stroke
27:1586-1591[Abstract/Free Full Text].
-
Huang A,
Huang PL,
Panahian N,
Dalkara T,
Fishman MC,
Moskowitz MA
(1994)
Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase.
Science
265:1883-1885[Abstract/Free Full Text].
-
Iadecola C
(1992)
Does nitric oxide mediate the increases in cerebral blood flow elicited by hypercapnia?
Proc Natl Acad Sci USA
89:3913-3916[Abstract/Free Full Text].
-
Iadecola C
(1997)
Bright and dark sides of nitric oxide in ischemic brain damage.
Trends Neurosci
20:132-138[Web of Science][Medline].
-
Iadecola C,
Zhang F
(1996)
Permissive and obligatory roles of NO in cerebrovascular responses to hypercapnia and acetylcholine.
Am J Physiol
271:R990-R1001[Abstract/Free Full Text].
-
Iadecola C,
Zhang F,
Xu X
(1995a)
Inhibition of inducible nitric oxide synthase ameliorates cerebral ischemic damage.
Am J Physiol
268:R286-R292[Abstract/Free Full Text].
-
Iadecola C,
Zhang F,
Xu X,
Casey R,
Ross ME
(1995b)
Inducible nitric oxide synthase gene expression in brain following focal cerebral ischemia.
J Cereb Blood Flow Metab
15:378-384[Web of Science][Medline].
-
Iadecola C,
Zhang F,
Casey R,
Clark HB,
Ross ME
(1996)
Inducible nitric oxide synthase gene expression in vascular cells following transient focal cerebral ischemia.
Stroke
27:1373-1380[Abstract/Free Full Text].
-
Koroshetz WJ,
Moskowitz MA
(1996)
Emerging treatments for stroke in humans.
Trends Pharmacol Sci
17:227-233[Medline].
-
Lin SZ,
Chiou AL,
Wang Y
(1996)
Ketamine antagonizes nitric oxide release from cerebral cortex after middle cerebral artery ligation in rats.
Stroke
27:747-752[Abstract/Free Full Text].
-
Lin TN,
He YY,
Wu G,
Khan M,
Hsu CY
(1993)
Effect of brain edema on infarct volume in a focal cerebral ischemia model in rats.
Stroke
24:117-121[Abstract/Free Full Text].
-
MacMicking JD,
Nathan C,
Hom G,
Chartrain N,
Fletcher DS,
Trumbauer M,
Stevens K,
Xie QW,
Sokol K,
Hutchinson N,
Chen H,
Mudgett JS
(1995)
Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase.
Cell
81:641-650[Web of Science][Medline].
-
Malinski T,
Bailey F,
Zhang ZG,
Chopp M
(1993)
Nitric oxide measured by a porphyrinic microsensor in rat brain after transient middle cerebral artery occlusion.
J Cereb Blood Flow Metab
13:355-358[Web of Science][Medline].
-
Marchal G,
Beaudouin V,
Rioux P,
de la Sayette V,
Le Doze F,
Viader F,
Derlon J-M,
Baron J-C
(1996)
Prolonged persistence of substantial volumes of potentially viable brain tissue after stroke.
Stroke
27:599-606[Abstract/Free Full Text].
-
Nagafuji T,
Sugiyama M,
Muto A,
Makino T,
Miyauchi T,
Nabata H
(1995)
The neuroprotective effect of a potent and selective inhibitor of type I NOS (L-MIN) in a rat model of focal cerebral ischaemia.
NeuroReport
6:1541-1545[Web of Science][Medline].
-
Nguyen T,
Brunson D,
Crespi CL,
Penman BW,
Wishnok JS,
Tannenbaum SR
(1992)
DNA damage and mutation in human cells exposed to nitric oxide in vitro.
Proc Natl Acad Sci USA
89:3030-3034[Abstract/Free Full Text].
-
Nogawa S,
Zhang F,
Ross E,
Iadecola C
(1997)
Cyclooxygenase-2 gene expression in neurons contributes to ischemic brain damage.
J Neurosci
17:2746-2755[Abstract/Free Full Text].
-
Nowicki JP,
Duval D,
Poignet H,
Scatton B
(1991)
Nitric oxide mediates neuronal death after focal cerebral ischemia in the mouse.
Eur J Pharmacol
204:339-340[Web of Science][Medline].
-
Ross ME,
Iadecola C
(1996)
Nitric oxide synthase expression in cerebral ischemia: neurochemical, immunocytochemical and molecular approaches.
In: Methods in enzymology (Packer L,
ed), pp 408-426. Orlando, FL: Academic.
-
Sessa A,
Perin A
(1994)
Diamine oxidase in relation to diamine and polyamine metabolism.
Agents Actions
43:69-77[Web of Science][Medline].
-
Siesjo BK
(1994)
Calcium-mediated processes in neuronal degeneration.
Ann NY Acad Sci
747:140-161[Web of Science][Medline].
-
Sobey CG,
Faraci FM
(1997)
Effects of a novel inhibitor of guanylyl cyclase on dilator responses of mouse cerebral arterioles.
Stroke
28:837-843[Abstract/Free Full Text].
-
Vodovotz Y,
Lucia MS,
Flanders KC,
Chesler L,
Xie QW,
Smith TW,
Weidner J,
Mumford R,
Webber R,
Nathan C,
Roberts AB,
Lippa CF,
Sporn MB
(1996)
Inducible nitric oxide synthase in tangle-bearing neurons of patients with Alzheimer's disease.
J Exp Med
184:1425-1433[Abstract/Free Full Text].
-
Wei XQ,
Charles IG,
Smith A,
Ure J,
Feng GJ,
Huang FP,
Xu D,
Muller W,
Moncada S,
Liew FY
(1995)
Altered immune responses in mice lacking inducible nitric oxide synthase.
Nature
375:408-411[Medline].
-
Yang G,
Chan PH,
Chen J,
Carlson E,
Chen SF,
Weinstein P,
Epstein CJ,
Kamii H
(1994)
Human copper-zinc superoxide dismutase transgenic mice are highly resistant to reperfusion injury after focal cerebral ischemia.
Stroke
25:165-170[Abstract].
-
Yoshida T,
Limmroth V,
Irikura K,
Moskowitz MA
(1994)
The NOS inhibitor, 7-nitroindazole, decreases focal infarct volume but not the response to topical acetylcholine in pial vessels.
J Cereb Blood Flow Metab
14:924-929[Web of Science][Medline].
-
Zhang F,
Iadecola C
(1992)
Stimulation of the fastigial nucleus enhances EEG recovery and reduces tissue damage after focal cerebral ischemia.
J Cereb Blood Flow Metab
12:962-970[Web of Science][Medline].
-
Zhang F,
Iadecola C
(1994)
Infarct measurement methodology.
J Cereb Blood Flow Metab
14:697-698[Web of Science][Medline].
-
Zhang J,
Dawson VL,
Dawson TM,
Snyder SH
(1994)
Nitric oxide activation of poly(ADP-ribose) synthetase in neurotoxicity.
Science
263:687-689[Abstract/Free Full Text].
-
Zhang ZG,
Reif D,
Macdonald J,
Tang WX,
Kamp D,
Gentile R,
Shakespeare W,
Murray R,
Chopp M
(1996)
ARL 17477, a potent and selective neuronal NOS inhibitor decreases infarct volume after transient middle cerebral artery occlusion in rats.
J Cereb Blood Flow Metab
16:599-604[Web of Science][Medline].
-
Zimmerman GA,
Meistrell MI,
Bloom O,
Cockroft KM,
Bianchi M,
Risucci D,
Broome J,
Farmer P,
Cerami A,
Vlassara H,
Tracey KJ
(1995)
Neurotoxicity of advanced glycation endproducts (AGEs) during focal stroke, and neuroprotective effects of aminoguanidine.
Proc Natl Acad Sci USA
92:3744-3748[Abstract/Free Full Text].
This article has been cited by other articles:
|
|
|
|
 
K. L. Lambertsen, B. H. Clausen, A. A. Babcock, R. Gregersen, C. Fenger, H. H. Nielsen, L. S. Haugaard, M. Wirenfeldt, M. Nielsen, F. Dagnaes-Hansen, et al.
Microglia Protect Neurons against Ischemia by Synthesis of Tumor Necrosis Factor
J. Neurosci.,
February 4, 2009;
29(5):
1319 - 1330.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Zhang, J. An, D. K. Strickland, and M. Yepes
The Low-Density Lipoprotein Receptor-Related Protein 1 Mediates Tissue-Type Plasminogen Activator-Induced Microglial Activation in the Ischemic Brain
Am. J. Pathol.,
February 1, 2009;
174(2):
586 - 594.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Muhammad, W. Barakat, S. Stoyanov, S. Murikinati, H. Yang, K. J. Tracey, M. Bendszus, G. Rossetti, P. P. Nawroth, A. Bierhaus, et al.
The HMGB1 Receptor RAGE Mediates Ischemic Brain Damage
J. Neurosci.,
November 12, 2008;
28(46):
12023 - 12031.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. An, C. Zhang, R. Polavarapu, X. Zhang, X. Zhang, and M. Yepes
Tissue-type plasminogen activator and the low-density lipoprotein receptor-related protein induce Akt phosphorylation in the ischemic brain
Blood,
October 1, 2008;
112(7):
2787 - 2794.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Kunz, T. Abe, K. Hochrainer, M. Shimamura, J. Anrather, G. Racchumi, P. Zhou, and C. Iadecola
Nuclear Factor-{kappa}B Activation and Postischemic Inflammation Are Suppressed in CD36-Null Mice after Middle Cerebral Artery Occlusion
J. Neurosci.,
February 13, 2008;
28(7):
1649 - 1658.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. W.T. Liu and P. L. Huang
Cardiovascular roles of nitric oxide: A review of insights from nitric oxide synthase gene disrupted mice
Cardiovasc Res,
January 1, 2008;
77(1):
19 - 29.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Zhang, R. Polavarapu, H. She, Z. Mao, and M. Yepes
Tissue-Type Plasminogen Activator and the Low-Density Lipoprotein Receptor-Related Protein Mediate Cerebral Ischemia-Induced Nuclear Factor-{kappa}B Pathway Activation
Am. J. Pathol.,
October 1, 2007;
171(4):
1281 - 1290.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Orio, A. Kunz, T. Kawano, J. Anrather, P. Zhou, and C. Iadecola
Lipopolysaccharide Induces Early Tolerance to Excitotoxicity via Nitric Oxide and cGMP
Stroke,
October 1, 2007;
38(10):
2812 - 2817.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Fogal, J. Li, D. Lobner, L. D. McCullough, and S. J. Hewett
System xc Activity and Astrocytes Are Necessary for Interleukin-1{beta}-Mediated Hypoxic Neuronal Injury
J. Neurosci.,
September 19, 2007;
27(38):
10094 - 10105.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. J. Kim, M. Rowe, M. Ren, J.-S. Hong, P.-S. Chen, and D.-M. Chuang
Histone Deacetylase Inhibitors Exhibit Anti-Inflammatory and Neuroprotective Effects in a Rat Permanent Ischemic Model of Stroke: Multiple Mechanisms of Action
J. Pharmacol. Exp. Ther.,
June 1, 2007;
321(3):
892 - 901.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Sunday, C. Osuna, D. N. Krause, and S. P. Duckles
Age alters cerebrovascular inflammation and effects of estrogen
Am J Physiol Heart Circ Physiol,
May 1, 2007;
292(5):
H2333 - H2340.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. A. Weissman, C. M. Sottas, P. Zhou, C. Iadecola, and M. P. Hardy
Testosterone production in mice lacking inducible nitric oxide synthase expression is sensitive to restraint stress
Am J Physiol Endocrinol Metab,
February 1, 2007;
292(2):
E615 - E620.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Pacher, J. S. Beckman, and L. Liaudet
Nitric Oxide and Peroxynitrite in Health and Disease
Physiol Rev,
January 1, 2007;
87(1):
315 - 424.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. L. Turgeon, M. C. Carr, P. M. Maki, M. E. Mendelsohn, and P. M. Wise
Complex Actions of Sex Steroids in Adipose Tissue, the Cardiovascular System, and Brain: Insights from Basic Science and Clinical Studies
Endocr. Rev.,
October 1, 2006;
27(6):
575 - 605.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Vagnerova, P. D. Hurn, A. Bhardwaj, and J. R. Kirsch
Sigma 1 receptor agonists act as neuroprotective drugs through inhibition of inducible nitric oxide synthase.
Anesth. Analg.,
August 1, 2006;
103(2):
430 - 4, table of contents.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Sunday, M. M. Tran, D. N. Krause, and S. P. Duckles
Estrogen and progestagens differentially modulate vascular proinflammatory factors
Am J Physiol Endocrinol Metab,
August 1, 2006;
291(2):
E261 - E267.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Schroeter, P. Zickler, D. T. Denhardt, H.-P. Hartung, and S. Jander
Increased thalamic neurodegeneration following ischaemic cortical stroke in osteopontin-deficient mice
Brain,
June 1, 2006;
129(6):
1426 - 1437.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. A. Kidd, H. Hong, A. Majid, D. I. Kaufman, and A. F. Chen
Inhibition of Brain GTP Cyclohydrolase I and Tetrahydrobiopterin Attenuates Cerebral Infarction via Reducing Inducible NO Synthase and Peroxynitrite in Ischemic Stroke
Stroke,
December 1, 2005;
36(12):
2705 - 2711.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Zhang, M. Komine-Kobayashi, R. Tanaka, M. Liu, Y. Mizuno, and T. Urabe
Edaravone Reduces Early Accumulation of Oxidative Products and Sequential Inflammatory Responses After Transient Focal Ischemia in Mice Brain
Stroke,
October 1, 2005;
36(10):
2220 - 2225.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Lazovic, A. Basu, H.-W. Lin, R. P. Rothstein, J. K. Krady, M. B. Smith, and S. W. Levison
Neuroinflammation and Both Cytotoxic and Vasogenic Edema Are Reduced in Interleukin-1 Type 1 Receptor-Deficient Mice Conferring Neuroprotection
Stroke,
October 1, 2005;
36(10):
2226 - 2231.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Mollace, C. Muscoli, E. Masini, S. Cuzzocrea, and D. Salvemini
Modulation of Prostaglandin Biosynthesis by Nitric Oxide and Nitric Oxide Donors
Pharmacol. Rev.,
June 1, 2005;
57(2):
217 - 252.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. R. Sunico, F. Portillo, D. Gonzalez-Forero, and B. Moreno-Lopez
Nitric Oxide-Directed Synaptic Remodeling in the Adult Mammal CNS
J. Neurosci.,
February 9, 2005;
25(6):
1448 - 1458.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. R. Madamanchi, A. Vendrov, and M. S. Runge
Oxidative Stress and Vascular Disease
Arterioscler. Thromb. Vasc. Biol.,
January 1, 2005;
25(1):
29 - 38.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E.-M. Park, S. Cho, K. Frys, G. Racchumi, P. Zhou, J. Anrather, and C. Iadecola
Interaction Between Inducible Nitric Oxide Synthase and Poly(ADP-ribose) Polymerase in Focal Ischemic Brain Injury
Stroke,
December 1, 2004;
35(12):
2896 - 2901.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. E. Baker, V. M. Brautigam, and J. J. Watters
Estrogen Modulates Microglial Inflammatory Mediator Production via Interactions with Estrogen Receptor {beta}
Endocrinology,
November 1, 2004;
145(11):
5021 - 5032.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W.-N. Qi, L.-E. Chen, L. Zhang, J. P. Eu, A. V. Seaber, and J. R. Urbaniak
Reperfusion injury in skeletal muscle is reduced in inducible nitric oxide synthase knockout mice
J Appl Physiol,
October 1, 2004;
97(4):
1323 - 1328.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Qi, T. Hosoi, Y. Okuma, M. Kaneko, and Y. Nomura
Sodium 4-Phenylbutyrate Protects against Cerebral Ischemic Injury
Mol. Pharmacol.,
October 1, 2004;
66(4):
899 - 908.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Iohom, S. Szarvas, V. Larney, J. O'Brien, E. Buckley, M. Butler, and G. Shorten
Perioperative Plasma Concentrations of Stable Nitric Oxide Products Are Predictive of Cognitive Dysfunction After Laparoscopic Cholecystectomy
Anesth. Analg.,
October 1, 2004;
99(4):
1245 - 1252.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Nakase, G. Sohl, M. Theis, K. Willecke, and C. C.G. Naus
Increased Apoptosis and Inflammation after Focal Brain Ischemia in Mice Lacking Connexin43 in Astrocytes
Am. J. Pathol.,
June 1, 2004;
164(6):
2067 - 2075.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Nurmi, P. J. Lindsberg, M. Koistinaho, W. Zhang, E. Juettler, M.-L. Karjalainen-Lindsberg, F. Weih, N. Frank, M. Schwaninger, and J. Koistinaho
Nuclear Factor-{kappa}B Contributes to Infarction After Permanent Focal Ischemia
Stroke,
April 1, 2004;
35(4):
987 - 991.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-S. Won, Y.-B. Im, L. Key, I. Singh, and A. K. Singh
The Involvement of Glucose Metabolism in the Regulation of Inducible Nitric Oxide Synthase Gene Expression in Glial Cells: Possible Role of Glucose-6-Phosphate Dehydrogenase and CCAAT/Enhancing Binding Protein
J. Neurosci.,
August 20, 2003;
23(20):
7470 - 7478.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Ding-Zhou, C. Marchand-Verrecchia, B. Palmier, N. Croci, P.-E. Chabrier, M. Plotkine, and I. Margaill
Neuroprotective Effects of (S)-N-[4-[4-[(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)carbonyl]-1-piperazinyl]phenyl]-2-thiophenecarboximid-amide (BN 80933), an Inhibitor of Neuronal Nitric-Oxide Synthase and an Antioxidant, in Model of Transient Focal Cerebral Ischemia in Mice
J. Pharmacol. Exp. Ther.,
August 1, 2003;
306(2):
588 - 594.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Lue, A. P. Sinha Hikim, C. Wang, A. Leung, and R. S. Swerdloff
Functional Role of Inducible Nitric Oxide Synthase in the Induction of Male Germ Cell Apoptosis, Regulation of Sperm Number, and Determination of Testes Size: Evidence from Null Mutant Mice
Endocrinology,
July 1, 2003;
144(7):
3092 - 3100.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Goyagi, T. J.K. Toung, J. R. Kirsch, R. J. Traystman, R. C. Koehler, P. D. Hurn, and A. Bhardwaj
Neuroprotective {kappa}-Opioid Receptor Agonist BRL 52537 Attenuates Ischemia-Evoked Nitric Oxide Production In Vivo in Rats
Stroke,
June 1, 2003;
34(6):
1533 - 1538.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Q. Li, Y. Guo, Y.-T. Xuan, C. J. Lowenstein, S. C. Stevenson, S. D. Prabhu, W.-J. Wu, Y. Zhu, and R. Bolli
Gene Therapy With Inducible Nitric Oxide Synthase Protects Against Myocardial Infarction via a Cyclooxygenase-2-Dependent Mechanism
Circ. Res.,
April 18, 2003;
92(7):
741 - 748.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Zhang, C. G. Looney, W.-N. Qi, L.-E. Chen, A. V. Seaber, J. S. Stamler, and J. R. Urbaniak
Reperfusion injury is reduced in skeletal muscle by inhibition of inducible nitric oxide synthase
J Appl Physiol,
April 1, 2003;
94(4):
1473 - 1478.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Vartiainen, G. Goldsteins, V. Keksa-Goldsteine, P. H. Chan, and J. Koistinaho
Aspirin Inhibits p44/42 Mitogen-Activated Protein Kinase and Is Protective Against Hypoxia/Reoxygenation Neuronal Damage
Stroke,
March 1, 2003;
34(3):
752 - 757.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Cherian, G. Chacko, C. Goodman, and C. S. Robertson
Neuroprotective Effects of L-Arginine Administration after Cortical Impact Injury in Rats: Dose Response and Time Window
J. Pharmacol. Exp. Ther.,
February 1, 2003;
304(2):
617 - 623.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Y. Zhu, S. H. Liu, H. S. Sun, and Y. M. Lu
Expression of Inducible Nitric Oxide Synthase after Focal Cerebral Ischemia Stimulates Neurogenesis in the Adult Rodent Dentate Gyrus
J. Neurosci.,
January 1, 2003;
23(1):
223 - 229.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Di Napoli, A.A. Taccardi, M. Oliver, and R. De Caterina
Statins and stroke: evidence for cholesterol-independent effects
Eur. Heart J.,
December 2, 2002;
23(24):
1908 - 1921.
[PDF]
|
|
|
|
|
|
|
L. Acarin, B. Gonzalez, and B. Castellano
Decrease of Proinflammatory Molecules Correlates With Neuroprotective Effect of the Fluorinated Salicylate Triflusal After Postnatal Excitotoxic Damage
Stroke,
October 1, 2002;
33(10):
2499 - 2505.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. S. Han, Y. Qiao, M. Karabiyikoglu, R. G. Giffard, and M. A. Yenari
Influence of Mild Hypothermia on Inducible Nitric Oxide Synthase Expression and Reactive Nitrogen Production in Experimental Stroke and Inflammation
J. Neurosci.,
May 15, 2002;
22(10):
3921 - 3928.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Krishnadasan, C. R. Hampton, J. Griscavage-Ennis, R. J. Dabal, and E. D. Verrier
Molecular Mechanisms of Neurologic Injury Following Cardiopulmonary Bypass
Seminars in Cardiothoracic and Vascular Anesthesia,
March 1, 2002;
6(1):
43 - 53.
[Abstract]
[PDF]
|
|
|
|
|
|
|
H. A. Arnett, R. P. Hellendall, G. K. Matsushima, K. Suzuki, V. E. Laubach, P. Sherman, and J. P.-Y. Ting
The Protective Role of Nitric Oxide in a Neurotoxicant- Induced Demyelinating Model
J. Immunol.,
January 1, 2002;
168(1):
427 - 433.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. B. Mackensen, M. Patel, H. Sheng, C. L. Calvi, I. Batinic-Haberle, B. J. Day, L. P. Liang, I. Fridovich, J. D. Crapo, R. D. Pearlstein, et al.
Neuroprotection from Delayed Postischemic Administration of a Metalloporphyrin Catalytic Antioxidant
J. Neurosci.,
July 1, 2001;
21(13):
4582 - 4592.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Goyagi, S. Goto, A. Bhardwaj, V. L. Dawson, P. D. Hurn, and J. R. Kirsch
Neuroprotective Effect of {{sigma}}1-Receptor Ligand 4-Phenyl-1-(4-Phenylbutyl) Piperidine (PPBP) Is Linked to Reduced Neuronal Nitric Oxide Production
Stroke,
July 1, 2001;
32(7):
1613 - 1620.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Saito, H. Kamii, I. Kato, S. Takasawa, T. Kondo, P. H. Chan, H. Okamoto, T. Yoshimoto, and R. L. Macdonald
Transgenic CuZn-Superoxide Dismutase Inhibits NO Synthase Induction in Experimental Subarachnoid Hemorrhage Editorial Comment
Stroke,
July 1, 2001;
32(7):
1652 - 1657.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Amin-Hanjani, N. E. Stagliano, M. Yamada, P. L. Huang, J. K. Liao, M. A. Moskowitz, C. Y. Hsu, and A. Nassief
Mevastatin, an HMG-CoA Reductase Inhibitor, Reduces Stroke Damage and Upregulates Endothelial Nitric Oxide Synthase in Mice Editorial Comment
Stroke,
April 1, 2001;
32(4):
980 - 986.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Mary, F. Wahl, A. Uzan, and J.-M. Stutzmann
Enoxaparin in Experimental Stroke : Neuroprotection and Therapeutic Window of Opportunity
Stroke,
April 1, 2001;
32(4):
993 - 999.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Iadecola, K. Niwa, S. Nogawa, X. Zhao, M. Nagayama, E. Araki, S. Morham, and M. E. Ross
Reduced susceptibility to ischemic brain injury and N-methyl-D-aspartate-mediated neurotoxicity in cyclooxygenase-2-deficient mice
PNAS,
January 30, 2001;
98(3):
1294 - 1299.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Fisher and W. Schaebitz
An Overview of Acute Stroke Therapy: Past, Present, and Future
Arch Intern Med,
November 27, 2000;
160(21):
3196 - 3206.
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Xu, L. He, S. H. Ahmed, S.-W. Chen, M. P. Goldberg, J. S. Beckman, C. Y. Hsu, and C. Iadecola
Oxygen-Glucose Deprivation Induces Inducible Nitric Oxide Synthase and Nitrotyrosine Expression in Cerebral Endothelial Cells Editorial Comment
Stroke,
July 1, 2000;
31(7):
1744 - 1751.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Iwase, K. Miyanaka, A. Shimizu, A. Nagasaki, T. Gotoh, M. Mori, and M. Takiguchi
Induction of Endothelial Nitric-oxide Synthase in Rat Brain Astrocytes by Systemic Lipopolysaccharide Treatment
J. Biol. Chem.,
April 14, 2000;
275(16):
11929 - 11933.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Castillo, R. Rama, and A. Davalos
Nitric Oxide-Related Brain Damage in Acute Ischemic Stroke
Stroke,
April 1, 2000;
31(4):
852 - 857.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. T. Akama and L. J. Van Eldik
beta -Amyloid Stimulation of Inducible Nitric-oxide Synthase in Astrocytes Is Interleukin-1beta - and Tumor Necrosis Factor-alpha (TNFalpha )-dependent, and Involves a TNFalpha Receptor-associated Factor- and NFkappa B-inducing Kinase-dependent Signaling Mechanism
J. Biol. Chem.,
March 10, 2000;
275(11):
7918 - 7924.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Lang-Lazdunski, K. Matsushita, L. Hirt, C. Waeber, J.-P. G. Vonsattel, M. A. Moskowitz, and W. D. Dietrich
Spinal Cord Ischemia : Development of a Model in the Mouse Editorial Comment: Development of a Model in the Mouse
Stroke,
January 1, 2000;
31(1):
208 - 213.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Bartholdy, A. Nansen, J. E. Christensen, O. Marker, and A. R. Thomsen
Inducible nitric-oxide synthase plays a minimal role in lymphocytic choriomeningitis virus-induced, T cell-mediated protective immunity and immunopathology
J. Gen. Virol.,
November 1, 1999;
80(11):
2997 - 3005.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
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]
|
|
|
|
|
|
|
M. van Lookeren Campagne, H. Thibodeaux, N. van Bruggen, B. Cairns, R. Gerlai, J. T. Palmer, S. P. Williams, and D. G. Lowe
Evidence for a protective role of metallothionein-1 in focal cerebral ischemia
PNAS,
October 26, 1999;
96(22):
12870 - 12875.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. J. Vaughan and N. Delanty
Neuroprotective Properties of Statins in Cerebral Ischemia and Stroke
Stroke,
September 1, 1999;
30(9):
1969 - 1973.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. O'MAHONY and M. J KENDALL
Nitric oxide in acute ischaemic stroke: a target for neuroprotection
J. Neurol. Neurosurg. Psychiatry,
July 1, 1999;
67(1):
1 - 3.
[Full Text]
|
|
|
|
|
|
|
S. I. Savitz and D. M. Rosenbaum
Review : Gene Expression after Cerebral Ischemia
Neuroscientist,
July 1, 1999;
5(4):
238 - 253.
[Abstract]
[PDF]
|
|
|
|
|
|
|
D. J. Lefer, S. P. Jones, W. G. Girod, A. Baines, M. B. Grisham, A. S. Cockrell, P. L. Huang, and R. Scalia
Leukocyte-endothelial cell interactions in nitric oxide synthase-deficient mice
Am J Physiol Heart Circ Physiol,
June 1, 1999;
276(6):
H1943 - H1950.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Iadecola, C. A. Salkowski, F. Zhang, T. Aber, M. Nagayama, S. N. Vogel, and M. Elizabeth Ross
The Transcription Factor Interferon Regulatory Factor 1 Is Expressed after Cerebral Ischemia and Contributes to Ischemic Brain Injury
J. Exp. Med.,
February 15, 1999;
189(4):
719 - 727.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Cho, B. T. Volpe, Y. Bae, O. Hwang, H. J. Choi, J. Gal, L. C. H. Park, C. K. Chu, J. Du, and T. H. Joh
Blockade of Tetrahydrobiopterin Synthesis Protects Neurons after Transient Forebrain Ischemia in Rat: A Novel Role for the Cofactor
J. Neurosci.,
February 1, 1999;
19(3):
878 - 889.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. A. Ratovitski, M. R. Alam, R. A. Quick, A. McMillan, C. Bao, C. Kozlovsky, T. A. Hand, R. C. Johnson, R. E. Mains, B. A. Eipper, et al.
Kalirin Inhibition of Inducible Nitric-oxide Synthase
J. Biol. Chem.,
January 8, 1999;
274(2):
993 - 999.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Yrjanheikki, R. Keinanen, M. Pellikka, T. Hokfelt, and J. Koistinaho
Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia
PNAS,
December 22, 1998;
95(26):
15769 - 15774.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Galea, S. B. Glickstein, D. L. Feinstein, E. V. Golanov, and D. J. Reis
Stimulation of cerebellar fastigial nucleus inhibits interleukin-1beta -induced cerebrovascular inflammation
Am J Physiol Heart Circ Physiol,
December 1, 1998;
275(6):
H2053 - H2063.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Shimizu-Sasamata, P. Bosque-Hamilton, P. L. Huang, M. A. Moskowitz, and E. H. Lo
Attenuated Neurotransmitter Release and Spreading Depression-Like Depolarizations after Focal Ischemia in Mutant Mice with Disrupted Type I Nitric Oxide Synthase Gene
J. Neurosci.,
November 15, 1998;
18(22):
9564 - 9571.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Nogawa, C. Forster, F. Zhang, M. Nagayama, M. E. Ross, and C. Iadecola
Interaction between inducible nitric oxide synthase and cyclooxygenase-2 after cerebral ischemia
PNAS,
September 1, 1998;
95(18):
10966 - 10971.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. M. Schwarz, B. Gierten, J.-P. Boissel, and U. Förstermann
Expressional Down-Regulation of Neuronal-Type Nitric Oxide Synthase I by Glucocorticoids in N1E-115 Neuroblastoma Cells
Mol. Pharmacol.,
August 1, 1998;
54(2):
258 - 263.
[Abstract]
[Full Text]
|
|
|
|
