Stroke. 1997;28:1283-1288
(Stroke. 1997;28:1283-1288.)
© 1997 American Heart Association, Inc.
Nitric Oxide Synthase in Models of Focal Ischemia
Amer F. Samdani, MD;
Ted M. Dawson, MD, PhD;
Valina L. Dawson, PhD
From the Departments of Neurology (A.F.S., T.M.D., V.L.D.), Neuroscience
(T.M.D., V.L.D.), and Physiology (V.L.D.), Johns Hopkins University School of
Medicine, Baltimore, Md.
Correspondence to Valina L. Dawson, PhD, Department of Neurology, Johns Hopkins University School of Medicine, 600 N Wolfe St, Path 2-210, Baltimore, MD 21287. E-mail: valina_dawson{at}qmail.bs.jhu.edu
 |
Abstract
|
|---|
Background and Purpose Cessation of blood flow to the
brain,
for even a few minutes, sets in motion a potential reversible
cascade
of events resulting in neuronal cell death. Oxygen free
radicals
and oxidants appear to play an important role in central
nervous
system injury after cerebral ischemia and reperfusion.
Recently,
divergent roles for the newly identified neuronal messenger
molecule
and oxygen radical, nitric oxide (NO), have been identified
in
various models of cerebral ischemia. Because of the chemical
and
physical properties of NO, the numerous
physiological activities
it mediates, and the lack
of specific agents to modulate the
activity of the different isoforms
of NO synthase (NOS), reports
regarding the role of NO in focal
cerebral ischemia have been
confounding and often conflicting.
Recent advances in pharmacology
and the development of transgenic
knockout mice specific for
the different isoforms of NOS have advanced
our knowledge and
clarified the role of NO in cerebral
ischemia.
Methods Animal models of focal ischemia employ
occlusion of nutrient cerebral vessels, most commonly the middle
cerebral artery. Primary cortical cultures are exposed to excitotoxic
or ischemic conditions, and the activities of NOS isoforms or
NO production are evaluated. Transgenic mice lacking expression
of either the neuronal isoform of NOS (nNOS), the
endothelial isoform of NOS (eNOS), or the immunologic
isoform of NOS (iNOS) have been examined in models of excitotoxic
injury and ischemia.
Results Excitotoxic or ischemic conditions
excessively activate nNOS, resulting in concentrations of NO
that are toxic to surrounding neurons. Conversely, NO generated from
eNOS is critical in maintaining cerebral blood flow and reducing
infarct volume. iNOS, which is not normally present in healthy
tissue, is induced shortly after ischemia and contributes to
secondary late-phase damage.
Conclusions Pharmacological and genetic approaches have
significantly advanced our knowledge regarding the role of NO and the
different NOS isoforms in focal cerebral ischemia. nNOS and
iNOS play key roles in neurodegeneration, while eNOS plays a prominent
role in maintaining cerebral blood flow and preventing neuronal injury.
Key Words: cerebral ischemia free radicals nitric oxide glutamates neurotoxins oxidants
 |
Introduction
|
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Loss of blood flow to
the brain results in neuronal injury due
to both the cessation of blood
flow leading to oxygen and nutrient
deprivation and the
initiation of a cascade of secondary mechanisms
(Figure

).
1 This neurotoxic cascade involves
derangements in normal metabolic
and
physiological functions as well as recruitment of
cell death
processes. Thus, both restoration of blood supply and
control
of secondary neurotoxic cascades are necessary to limit
ischemic
neuronal damage. Numerous transmitter and second
messenger pathways
are inappropriately activated after the
initial ischemic event.
2 A major pathway leading
toward neuronal injury involves elevation
of extracellular glutamate
and activation of glutamate receptors,
with a subsequent increase in
intracellular calcium, resulting
in generation of free radicals and
NO.
3 Glutamate initiates
its actions postsynaptically by
binding to four major types
of receptors: metabotropic receptors, NMDA
receptors, AMPA receptors,
and kainate receptors. NMDA receptor
activation mediates, in
large part, glutamate excitotoxicity and
neuronal damage after
focal ischemia.
4
Glutamate-stimulated NMDA receptors flux calcium
and activate a
variety of intracellular calciumdependent
enzymes and processes, of
which activation of neuronal NOS (nNOS)
plays a prominent role. Thus,
overproduction of NO from excessive
or inappropriate
stimulation of nNOS appears to mediate a major
component of excitotoxic
damage.

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Figure 1. Postulated mechanisms of neuronal death after focal
ischemia. CBF indicates cerebral blood flow; VSCC,
voltage-dependent calcium channels; NMDA-R,
N-methyl-D-aspartate receptor; PLA2,
phospholipase A2; PKC, protein kinase C; CaMK II,
calcium-calmodulindependent protein kinase II; ROS, reactive oxygen
species, and ICE, interleukin-1ß converting enzyme.
|
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NOS Isoforms
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There are three NOS isoforms that are named after the tissue
from
which they were first cloned and numbered in the order
in which they
were cloned
5 6 (Table 1

). nNOS (type I) and
endothelial
NOS (eNOS) (type III) are constitutively
expressed and are calcium
dependent. Immunologic NOS (iNOS) (type II)
is expressed after
immunologic challenge and neuronal injury and is
calcium independent
under most circumstances.
7
NOS catalyzes the stoichiometric conversion of
L-arginine to NO and citrulline in the presence of oxygen
and NADPH. There are multiple sites on all three isoforms for cofactor
and substrate interactions that provide potential targets for
pharmacological regulation of NOS catalytic
activity.7 8
 |
nNOS Mediates Early Neuronal Injury
|
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nNOS is primarily expressed in a small population of neurons
throughout
the nervous system and a variety of other tissues throughout
the
body (Table 1

). It is neuronally produced NO that is believed
to
mediate synaptic plasticity and neuronal signaling and, after
ischemic
damage, neurotoxicity.
In primary cortical neuronal cultures, in which nNOS is expressed at
levels equivalent to in vivo expression, glutamate and NMDA
neurotoxicity are mediated largely by excess formation of NO.
Coexposure of primary cortical neurons to NMDA and arginine analogue
NOS inhibitors, flavoprotein inhibitors,
calmodulin antagonists or agents that bind
calmodulin, and calcineurin inhibitors, all of
which decrease NOS catalytic activity, results in neuroprotection
against NMDA neurotoxicity.3 7 9 NO is toxic and appears
to activate death pathways similar to those activated
by NMDA, since compounds that directly release NO are neurotoxic and
exhibit the same kinetics of death as NMDA neurotoxicity. Elimination
of nNOS through transgenic technology results in primary neuronal
cultures that are resistant to NMDA neurotoxicity, indicating
that nNOS neurons are the source of neurotoxic NO.10
Neuronal injury after combined oxygen-glucose deprivation
of neuronal cultures, which may more accurately reflect in vivo
ischemic conditions, is reversed by NOS inhibitors.
Additionally, neuronal cultures from nNOS null transgenic mice are
markedly resistant to combined oxygen-glucose
deprivation compared with wild-type
cultures.10 These in vitro studies indicate that NO is not
only a major mediator of glutamate excitotoxicity but also contributes
in a significant way to the neuronal damage after oxygen-glucose
deprivation. While these observations have been replicated
in a variety of cell culture systems as well as slice preparations,
there are conflicting reports in the literature in which NO-mediated
neurotoxicity was not observed.3 7 9 In many cases these
differences can be attributed to insufficient expression of nNOS or
differences in experimental paradigms altering the reaction pathways of
NO. For instance, recent studies indicate that an NO component to NMDA
neurotoxicity is critically dependent on the number of nNOS neurons and
the level of nNOS protein.11 The expression of nNOS is
critically dependent on the culture condition used, in that neurons
grown on glial feeder layers contain relatively low levels of nNOS,
whereas neurons grown on a polyornithine matrix tend to contain high
levels of nNOS. A variety of neurotrophins markedly increase the number
of nNOS neurons, nNOS protein, and NOS catalytic activity and enhance
NMDA neurotoxicity through NO-dependent mechanisms when neurons are
grown on glial feeder layers. In contrast, when rat or mouse primary
cortical neurons are grown on a polyornithine matrix, neurotrophins
have no influence on nNOS neuronal number or NOS catalytic activity and
reduce NMDA neurotoxicity. Primary neuronal cultures from mice lacking
nNOS grown on a glial feeder layer fail to respond to
neurotrophin-mediated enhancement of neurotoxicity. Thus, nNOS
expression and NMDA, NO-mediated neurotoxicity are dependent on the
culture paradigm, and neurotrophins regulate the susceptibility to NMDA
neurotoxicity through modulation of nNOS.
In animal models of focal ischemia, inhibition of nNOS with
concentrations of NOS inhibitors that do not perturb eNOS
activity reduces infarct volume after MCA occlusion in mice, rats, and
cats.12 13 14 Additionally, selective nNOS
inhibitors, including 7-nitroindazole15 16 and
ARL 17477,17 which do not influence eNOS activity but
effectively diminish nNOS activity in vivo, are consistently
neuroprotective in models of focal ischemia. Although many
investigators have observed a reduction in infarct volume with a
variety of nonselective NOS inhibitors, other investigators
have observed either no effect or exacerbation of injury. Exacerbation
of injury seems to occur at higher doses of nonselective NOS
inhibitors through inhibition of eNOS, resulting in
deleterious alterations of cerebral blood flow and subsequent increased
infarction volume.12 13 14
Clarification of the role of the different NOS isoforms in focal
ischemia was obtained from transgenic mice studies in mice
lacking nNOS, eNOS, or iNOS (Table 2
). Despite the
usefulness of these models, one needs to be cognizant that transgenic
animals have lacked the respective enzyme since birth and that
compensatory processes may have occurred that enabled the animal to
reach adulthood. It is conceivable that these compensatory processes
account for phenotypic differences in transgenic versus wild-type mice.
Thus, it is important to recognize these potential limitations and
confirm observations obtained with the use of transgenic technology
with conventional pharmacological approaches.
After permanent focal MCA occlusion, nNOS null mice have reduced
infarct volumes compared with age-matched wild-type
controls.18 The reduction of infarct volume in nNOS null
transgenic mice could be reversed by administration of nonspecific NOS
inhibitors at concentrations sufficient to inhibit
NO-dependent relaxation of pial vessels. Under these conditions,
infarct volumes were equivalent to those of wild-type mice. Genetic
deletion of nNOS also conferred dramatic resistance to focal
ischemic injury in a reperfusion model of transient MCA
occlusion,19 and hippocampal damage was reduced in a model
of global cerebral ischemia.20 Selective nNOS
inhibitors may need to be used cautiously, since inhibition
of nNOS can activate nuclear factor-
B (NF-
B), leading to
induction of iNOS, which could exacerbate neuronal injury in the later
stages following cerebral ischemia.21
Although nNOS is constitutively expressed, after certain pathological
insults nNOS can be induced in some cells through new protein
synthesis. For instance, after MCA occlusion in the rat, a rapid
upregulation of nNOS mRNA as well as nNOS protein and
NADPH-diaphorase positive staining has been observed in the
ischemic lesion.22 It is possible that the
increase in nNOS expression contributes to the spread of neuronal
damage after ischemic injury. However, recent studies indicate
that upregulation of nNOS may subserve a restorative function through
NO's activation of the Ras extracellular signalregulated protein
kinase pathway, leading to long-term changes in neuronal
plasticity.23
Anatomic localization of nNOS reveals a distribution for the enzyme
that does not correlate with any one neurotransmitter but is coincident
with the histochemical stain
NADPH-diaphorase.24 25
NADPH-diaphorasepositive neurons are of extreme interest
because they are relatively spared from neuronal cell death after
vascular stroke and excitotoxicity and in Huntington's and
Alzheimer's diseases.26 Recent studies suggest
that the mitochondrial Mn-SOD accounts for the selective resistance of
nNOS neurons to toxic insults.27 nNOS neurons are
selectively enriched in Mn-SOD. Antisense knockdown of Mn-SOD renders
nNOS neurons susceptible to NMDA neurotoxicity but does not influence
the overall susceptibility of non-nNOS cortical neurons to NMDA
toxicity. Furthermore, overexpression of Mn-SOD by adenoviral-mediated
gene transfer provides dramatic protection against NMDA and NO toxicity
in cortical cultures.
iNOS Contributes to Late Neuronal Injury
iNOS is not detectable in healthy tissue (Table 1
). In addition to
macrophages and microglia, iNOS under pathological conditions
can be expressed in most tissues, including neurons, astrocytes, and
endothelial cells.28 Induction of iNOS in
vitro results in delayed neuronal cell death29 30 31 and can
also exacerbate glutamate excitotoxicity.32 In human
disease, iNOS expression may play a role in demyelination in multiple
sclerosis33 and may contribute to neuronal injury in
severe AIDS dementia.34 In rat brain, iNOS protein and
catalytic activity are detectable 12 hours after cerebral
ischemia, peak at 48 hours, and return to baseline in 7
days.35 The relatively selective iNOS
inhibitor aminoguanidine, administered 24 hours after the
ischemic insult, results in reduced infarct volumes compared
with vehicle-treated controls.36 Neuroprotection by
aminoguanidine is reversed by excess substrate L-arginine
but not the inactive stereoisomer D-arginine. Clarification
of the role of iNOS in late neuronal injury accompanying cerebral
ischemia is provided by the observation that mice lacking the
gene for iNOS have significantly reduced infarct volumes compared with
wild-type controls (Table 2
).37 Whether upregulation of
iNOS in endothelial cells could play a protective role
after stroke through beneficial effects on cerebral blood flow is not
known.
 |
eNOS Is Neuroprotective
|
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Attempts to understand the vasodilatory effects of organic
nitrates
and the mechanisms of acetylcholine-induced relaxation of
vascular
smooth muscle led to the discovery that
endothelially derived
NO is critical in the regulation
of vascular hemodynamics.
38 eNOS is
constitutively expressed and briefly activated by increases
in
intracellular calcium (Table 1

). In addition to expression
in
endothelial cells, a small population of neurons in the
central
nervous system express eNOS.
39
NO is a major regulator of vascular hemodynamics and is
the primary messenger molecule mediating blood vessel
relaxation.40 Even partial inhibition of eNOS can result
in large changes in cerebral blood flow. In initial reports examining
ischemic outcomes after inhibition of NOS, investigators based
their conclusions regarding the role of NO in cerebral ischemia
on results obtained with nonspecific NOS
inhibitors.12 13 14 These nonselective agents
inhibit both eNOS and nNOS. Administration of nonspecific NOS
inhibitors results in constriction of pial arterioles,
reduction in cerebral blood flow, and subsequent increases in infarct
volume. In contrast, NO donors or intra-arterial
administration of L-arginine increases regional cerebral
blood flow and decreases infarct volume distal to MCA occlusion in
rats.12 13 14 Consistent with the notion that eNOS
subserves a protective role in cerebral ischemia by maintaining
regional cerebral blood flow is the observation that transgenic mice
that lack eNOS experience increased infarct volumes after MCA occlusion
(Table 2
).41 Furthermore, the nonspecific NOS
inhibitor, nitro-L-arginine, decreases the
infarct size in eNOS null mice but does not affect infarct size in
wild-type mice. The only target for nitro-L-arginine in
eNOS null mice during the acute phase is nNOS, and therefore these
studies highlight the dual actions of NO in focal ischemia.
Overproduction of NO from activation of nNOS leads to
neurotoxicity; however, production of NO from eNOS protects
brain tissue by maintaining regional cerebral blood flow. The
development of selective pharmacological tools and the development of
transgenic mice have allowed the dissection of the divergent roles for
NO in focal ischemia (Table 2
). eNOS protein and catalytic
activity, like nNOS, are also upregulated during the acute phase of
ischemia.42 Perhaps upregulation of eNOS subserves
a protective role by facilitating the maintenance of cerebral
blood flow in the setting of ischemia.
 |
Targets of NO
|
|---|
The exact pathways by which excessive NO formation results in
neuronal
cell death are not known. Disruption of any of the normal
physiological
processes mediated by NO could have
deleterious effects on neuronal
survival. Because of an unpaired
electron, NO is by definition
a free radical. The best known biological
targets of NO include
oxygen, transition metals,
iron-sulfurcontaining proteins,
and heme-containing
proteins
43 (Table 3

). An important and
well-studied
transition metal target of NO is the iron in the heme
moiety
of guanylate cyclase. NO alters the conformation of
guanylate
cyclase, which leads to the formation of cGMP.
cGMP plays little
if any role in the toxicity of NO; instead, it may be
neuroprotective.
44 NO readily reacts with several
iron-sulfur clustercontaining
proteins, including the mitochondrial
NADH-ubiquinone oxidoreductase
and NADH:succinate oxidoreductase. NO
inhibits both enzymes,
which may contribute to neuronal injury by
inhibiting oxidative
phosyphorylation.
7 NO also inhibits
cis-aconitase, another
iron-sulfur clustercontaining
protein, leading to inhibition
of glycolysis, and it reversibly
inhibits mitochondrial respiration
by competing with oxygen at
cytochrome oxidase.
45 NO reacts
with the thiols of many
proteins, resulting in
S-nitrosylation.
NO inhibits creatine
kinase activity through nitrosothiol modification.
46
Inhibition of creatine kinase leads to decrement in ATP due
to the
inhibition of phosphoryl transfer between phosphocreatine
and ATP,
which could contribute to neuronal injury by decreasing
the
availability of ATP. An established pathway of NO-mediated
neuronal
cell death is NO activation of the nuclear enzyme PARS.
47
NO activates PARS by damaging DNA. PARS catalyzes the transfer
of
ADP-ribose units from NAD to nuclear proteins. For every mole
of
ADP-ribose transferred, one mole of NAD is consumed, and
four free
energy equivalents of ATP are necessary to regenerate
NAD. Therefore,
overactivation of PARS can rapidly deplete cellular
energy stores. If
mitochondrial enzymes are simultaneously impaired
from
exposure to NO, the cell's ability to replace NAD and ATP
is
compromised, leading to energy failure and cell death (Figure

).
Recently,
targeted disruption of PARS provided compelling evidence for
participation
of PARS in NO-mediated toxicity.
48 Mutant
mouse islet cells
lacking PARS do not show DNA damageinduced NAD
depletion
and are more resistant to NO toxicity. However,
NO-mediated
cell death was not completely abolished in the mutant mice
islet
cells, suggesting the existence of alternative pathways for
NO-mediated
toxicity not involving PARS-mediated NAD depletion.
In biological systems, the most permissive reaction of NO is with
O2·- to produce the potent
oxidant ONOO-. Some investigators have stressed that
direct toxic effects of NO are modest49 and that NO may
have neuroprotective properties.13 Recent investigations
suggest that the majority of the toxic effects attributed to NO, as
well as O2·-, are due to
ONOO-.50 Interestingly, nNOS in the setting
of decreased substrate L-arginine availability, which would
occur during ischemia, is capable of producing both NO and
O2·-.51
Although both transgenic mice that lack nNOS18 and
transgenic mice that overexpress the
O2·- cytosolic scavenging
enzyme Cu2+/Zn-SOD52 have reduced infarct
volumes after permanent MCA occlusion, double transgenic mice lacking
nNOS and overexpressing Cu2+/Zn-SOD have significantly
smaller infarct volumes then either genetic manipulation
alone.53 Thus, it is the combination of the
O2·- produced
simultaneously with NO leading to ONOO-
formation that accounts for the majority of toxicity after cerebral
ischemia. Activation of NMDA receptors mediates the majority of
damage in focal ischemia. Interestingly, NMDA, but not
non-NMDA, receptor activation increases mitochondrial reactive oxygen
species,54 and NMDA, but not non-NMDA, receptor activation
is the major stimulator of nNOS activity. Perhaps it is through the
simultaneous production of NO and
O2·- that the majority of
the neurotoxic actions of glutamate are mediated. One of the major
targets of ONOO- is mitochondrial Mn-SOD. Thus,
ONOO- inactivation of Mn-SOD could initiate a
self-propagating cascade of neural injury through failure of
mitochondrial scavenging of
O2·-.55
NO-mediated cell death occurs through both necrotic and
apoptotic cell death pathways. Although NMDA-mediated neuronal
death is thought to be largely necrotic, the mode of cell death may
depend on the intensity of toxic insults of NMDA and NO and
mitochondrial function.56 57 In cortical cultures, mild
excitotoxic or free radical insults lead to delayed neuronal death
dominated by apoptotic features, whereas intense exposure to a
high concentration of NMDA or ONOO- induces necrotic cell
death. Activation of iNOS in primary neuronal cultures causes a slowly
progressive neuronal cell death that is dominated by apoptotic
features (K. Kopnisky et al, unpublished data, 1997). Recent studies in
nonneuronal systems suggest that iNOS-mediated toxicity occurs through
activation of interleukin-1ß converting enzymelike proteases and
P53-dependent pathways.58
 |
Conclusions
|
|---|
It is now clear that NO plays major roles in modulating brain
injury
after ischemic events. The development of selective
pharmacological
tools as well as the development of transgenic mice
lacking
each NOS isoform has greatly advanced our understanding of the
diverse
roles of NO in the central nervous system as well as the roles
of
NO in response to ischemic injury. Current studies indicate
that
NO plays a dual role in focal cerebral ischemia. Depending
on
its source, NO may be toxic or protective to the brain under
ischemic
conditions. Overproduction of NO from either
nNOS or iNOS leads
to neurotoxicity; however, NO production
from endothelial NOS
protects brain tissue by
maintaining regional cerebral blood
flow. These studies emphasize the
necessity of developing truly
selective inhibitors for nNOS
and iNOS to adequately protect
the brain from ischemic injury
due to overproduction of NO yet
simultaneously
maintain or enhance regional cerebral blood flow.
 |
Selected Abbreviations and Acronyms
|
|---|
| AMPA |
= |
-amino-3-hydroxy-5-methyl-4-isoxazolepropionate |
| eNOS |
= |
endothelial NOS |
| iNOS |
= |
immunologic NOS |
| MCA |
= |
middle cerebral artery |
| NMDA |
= |
N-methyl-D-aspartate |
| nNOS |
= |
neuronal NOS |
| NO |
= |
nitric oxide |
| NOS |
= |
nitric oxide synthase |
| O2·- |
= |
superoxide anion |
| ONOO- |
= |
peroxynitrite |
| PARS |
= |
poly(ADP-ribose) synthetase |
| SOD |
= |
superoxide dismutase |
|
 |
Acknowledgments
|
|---|
This study was supported in part by US Public Health Service
grants
NS 33277 and NS 01578 and by the International Life Sciences
Institute
(T.M.D.), US Public Health Service grant NS 33142, and the
American
Heart Association (V.L.D.). The authors thank Ann Schmidt for
secretarial
assistance and our colleagues who provided preprints of
their
work. Under an agreement between the Johns Hopkins University
and
Guilford Pharmaceuticals, T.M.D. and V.L.D. are entitled
to a share of
sales royalty received by the University from
Guilford Pharmaceuticals.
T.M.D. and the University also own
Guilford stock, which is subject to
certain restrictions under
University policy. The terms of this
arrangement have been reviewed
and approved by the University in
accordance with its conflict
of interest policies.
Received January 21, 1997;
revision received April 9, 1997;
accepted April 10, 1997.
 |
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