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J Neurol Neurosurg Psychiatry 1999;67:1–3
EDITORIAL
Nitric oxide in acute ischaemic stroke: a target for
neuroprotection
Stroke illness remains the single greatest cause of serious
physical disability in western countries and is a major con-
sumer of scarce medical resources. In the United
Kingdom, it is estimated that the management of stroke
patients accounts for 4%–5% of the National Health Serv-
ice budget.1 Clearly, any intervention that reduces cerebral
infarct size will have a major impact on the personal and
health budget costs of stroke illness. At present, low dose
aspirin and early referral to a dedicated “stroke unit” seem
to be the two positive things that physicians can do for
patients with acute ischaemic stroke that significantly
reduce mortality and dependency levels, for which there is
a firm evidence base. Treatment with thrombolytic drugs
may be beneficial in those with acute stroke who are fortu-
nate enough to obtain treatment within 3 hours of onset of
symptoms.2 However, with such a narrow therapeutic time
window, early thrombolysis is applicable in only a small
minority of patients. Similarly, human trials of several neu-
roprotective agents which work in animals have so far
proved disappointing.
In the midst of this therapeutic gloom, scientists and cli-
nicians have been forced to re-evaluate the potential thera-
peutic targets in the cerebral ischaemic cascade. Perhaps
the reason for the failure of neuroprotective interventions is
that trials have concentrated on inhibiting the early
components of the cascade which are triggered within
minutes of onset of ischaemia. Attempts to block these
processes after several hours, particularly glutamate activa-
tion of N-methyl-D-aspartate receptors and intracellular
calcium influx may be a futile exercise. Or, it may be that
potentially beneficial drugs simply do not reach their site of
action because of the ischaemia aVecting the penumbral
zone during the putative therapeutuc time window. Is there
then scope for eVective therapeutic intervention beyond
the first few hours of onset of ischaemia? Given recent evi-
dence indicating that the ischaemic penumbra in humans
remains metabolically viable for up to 24 hours after onset
of cerebral ischaemia,3 penumbral salvage and infarct vol-
ume reduction should, in theory, be feasible. Information
about the delayed neurodestructive pathways has increased
rapidly in recent years. Free radical oxidative damage, cell
energy depletion, and altered gene expression leading to
apoptotic cell death seem to be particularly important
processes in penumbral deterioration. The purpose of this
article is to focus on nitric oxide release as a delayed neu-
rodestructive mechanism in acute cerebral ischaemia and
the pharmacological methods of inhibiting it that may be
applicable in humans.
1
Nitric oxide and other oxidative free radicals in
cerebral ischaemia
By virtue of their unpaired electron, oxidative free radicals
(for example, hydroxyl and superoxide anions) are highly
reactive chemical species. Under normal circumstances,
free radicals that are produced as byproducts of cellular
metabolism are removed by free radical scavenging
enzymes, such as superoxide dismutase, catalase, glutath-
ione peroxidase, and other scavenging substances such as
á-tocopherol and ascorbic acid. Free radicals are produced
in high concentration after mitochondrial failure from cel-
lular deprivation of oxygen and glucose. Scavenger systems
become rapidly saturated, allowing free radicals to react
avidly with and damage complex nucleic acid, lipid, carbo-
hydrate, and protein molecules. This leads to major
disruption of normal cellular processes and eventual cell
death. Further, oxidative free radical release occurs on
reperfusion of an ischaemic area of brain, contributing to
socalled “reperfusion injury”. The importance of free radi-
cal cytotoxicity is further seen in transgenic mice underex-
pressing the superoxide dismutase gene. These animals
develop significantly larger cerebral infarcts than wild-type
mice.4 Conversely, intravascular administration of superox-
ide dismutase bound to polyethylene glycol results in
smaller cerebral infarcts in mice subjected to ischaemia-
reperfusion compared with vehicle treated animals.5 Also,
mice genetically altered to overexpress superoxide dis-
mutase develop significantly smaller cerebral infarcts.6
Superoxide dismutase-polyethylene glycol complex might
be beneficial in acute ischaemic stroke, given its eYcacy in
patients with severe head injury,7 but clinical trials in stroke
are awaited.
Nitric oxide is a free radical substance with several well
established physiological properties including functional
regulation of vascular smooth muscle cells, neurons, mac-
rophages, platelets, and neutrophils when produced in very
small, regulated concentrations. Nitric oxide synthase
activity and nitric oxide release are greatly increased in the
acutely ischaemic brain.8 Nitric oxide reacts rapidly with
superoxide to form peroxynitrite, which is a powerful oxi-
dant and highly cytotoxic (figure). Nitric oxide in high
concentration is indirectly neurotoxic through other
mechanisms including iron-mediated lipid peroxidation
(nitric oxide liberates iron from cell stores) and cell energy
depletion by disruption of mitochondrial enzymes and
nucleic acids. Release of nitric oxide may also trigger neu-
ronal apoptotic cell death.9
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2 O’Mahoney, Kendall

Acute cerebral ischaemia synthase (nNOS), leading to release of nitric oxide in high
local concentrations with resultant free radical damage.
Neuronal cell energy failure Studies involving transgenic mouse models and selective
Activation of microglial cells
nNOS inhibitors further highlight the importance of nNOS
in the pathophysiology of cerebral ischaemia (table). Huang
Depolarisation
et al11 have recently shown an average infarct volume reduc-
tion of 38% in nNOS knockout mice compared with
Glutamate release
wild-type mice. Similarly, treatment with selective inhibitors
Cytokine release of nNOS, 7-nitroindazole and ARL17477, produces signifi-
NMDA/Ca
2+
complex cant infarct volume reduction in rats (up to 27% with
7-nitroindazol.12 From these studies, it would seem that
Intracellular Ca
2+
influx enhancement of eNOS activity using nitric oxide donors
Microglial iNOS induction
combined with selective nNOS inhibitors is an appropriate
therapeutic strategy for ischaemic stroke in humans.
nNOS upregulation

NO* + O2* Inducible nitric oxide synthase in cerebral ischaemia

ONOO–
Lipid peroxidation Mitochondrial enzyme damage
Cell membrane Cell energy failure
Breakdown
CELL DEATH
Causes and consequences of nitric oxide release in acute cerebral ischaemia.
NMDA/Ca2+=N-methyl-D-aspartate/calcium ion membrane complex;
nNOS=neuronal nitric oxide synthase; iNOS=inducible nitric oxide
synthase; NO*=nitric oxide; O2*=superoxide anion;
ONOO=peroxynitrite.
Endothelial and neuronal nitric oxide synthase
isoforms in cerebral ischaemia
Nitric oxide release in acute cerebral ischaemia may have
positive as well as negative eVects (table). Increased
endothelial nitric oxide synthase (eNOS) activity seems to
be neuroprotective, probably through its cerebral vasodila-
tory eVects as well as by inhibition of platelet aggregation
and leucocyte endothelial adhesion. Mouse models of
stroke in which the eNOS gene is not expressed (eNOS
knockout) develop significantly larger cerebral infarcts
after permanent middle cerebral artery occlusion
(MCAO).10 Conversely, enhanced eNOS mediated nitric
oxide release with intravascular eNOS substrate,
L-arginine and nitric oxide donor drugs such as sodium
nitroprusside results in smaller cerebral infarcts than those
in vehicle treated animals.9
By contrast with the neuroprotective eVect of enhanced
endothelial nitric oxide, neuronal release of nitric oxide plays
a major part in ischaemic neurotoxicity. After cerebral
ischaemia, glutamate release in high concentration activates
several postsynaptic glutamate receptor/ion channel com-
plexes, most notably N-methyl-D-aspartate (NMDA)/Ca2+.
Intracellular Ca2+ influx activates several calcium dependent
cytodestructive enzymes, including neuronal nitric oxide
The situation is not quite as straightforward as this, however.
In rodents, the neuroprotective eVect of L-arginine and
nitric oxide donor drugs is lost about 2 hours after onset of
cerebral ischaemia. A greater cause for concern is the finding
DNA strand breaks
DNA base deamination
that intravascular L-arginine is associated with increased
infarct volume when its administration is delayed by 24
Apoptosis hours in rats.13 These time dependent opposite eVects of
L-arginine are thought to result from the appearance in the
ischaemic penumbra of the third nitric oxide synthase
isoform, inducible or immunological NOS (iNOS), after a
time lag of 6–12 hours (table).9 Inducible NOS is known to
contribute to delayed neuronal injury in stroke, as iNOS
knockout mice develop significantly smaller cerebral infarcts
after focal ischaemia than wild type mice.14 Inducible NOS
diVers fundamentally from eNOS and nNOS as its
activation is not calcium dependent—that is, iNOS may be
fully activated at basal intracellular concentrations of
calcium, whereas eNOS and nNOS only become upregu-
lated at high calcium concentrations.15 The appearance of
iNOS after cerebral ischaemia is delayed because, unlike
eNOS and nNOS, iNOS is not a constitutive enzyme and is
only produced after cytokine stimulation of neutrophils
resulting from local ischaemia.9 Inducible NOS messenger
RNA is detectable 12 hours after ischaemia, reaches peak
concentrations at 48 hours, and takes about 7 days to return
to baseline.16 The major source of delayed nitric oxide
release in the ischaemic brain, therefore, is thought to be
iNOS. The delayed appearance of iNOS would also explain
the dual early neuroprotective and later neurotoxic action of
L-arginine, as L-arginine is the principal natural substrate
for each of the three NOS isoforms.
Neuronal nitric oxide synthase as a therapeutic
target in cerebral ischaemia
Neuroprotection from selective nNOS inhibitors in
rodents seems to come from early intervention—that is, in
the first 2 hours after focal cerebral ischaemia. In a mouse
model of traumatic brain injury, the selective nNOS

Nitric oxide synthase isoforms and their pharmacological properties in relation to acute cerebral ischaemia

Time to protein,
catalytic EVect of NOS EVects of cerebral
Role in acute upregulation and Pharmacological antagonist on acute ischaemia on gene
NOS isoform Cell source Physiological function cerebral ischaemia9 peak antagonist cerebral ischaemia knockout mice

Endothelial Vascular Regulation of tissue Neuroprotective 1 hour, 24 hours - L-nitroarginine, Increased cerebral Increased cerebral
(eNOS) Endothelium perfusion - Nitro-L-arginine infarct volume9 infarct volume10
Mono-methyl
Ester (L-NAME)
Neuronal Neurons Mediation of Neurodestructive 10 minutes, 3 7-nitroindazole Decreased cerebral Decreased cerebral
(nNOS) synaptic plasticity hours infarct volume12 infarct volume11
and neuronal
signalling
Immunological Macrophages Oxidative free radical Neurodestructive 12 hours, 48 hours Aminoguanidine Decreased cerebral Decreased cerebral
or inducible Microglia destruction of infarct volume13 21–23 infarct volume14
(iNOS) Neutrophils infectious agents
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Nitric oxide in acute ischaemic stroke
inhibitor 7-nitroindazole significantly reduced the neuro-
logical deficit when given at 5, 30, and 60 minutes after
injury but not at 2 hours.17 Neuronal NOS has been shown
to have a major role in the cerebral hyperaemic response to
hypoxia18 and therefore inhibition of nNOS could, in
theory, disrupt an important compensatory mechanism in
cerebral ischaemia. In addition, there may be risks from
inhibiting an enzyme that contributes to such major
neurological functions as synaptic plasticity and neuronal
signalling. There is also concern that the finding of
disturbed, aggressive behaviour in nNOS knockout mice19
could mean that comparable adverse eVects with selective
nNOS inhibitors may occur in humans. Finally, inhibition
of nNOS activates nuclear factor-êâ that leads to induction
of iNOS,20 which could, in theory, indirectly increase tissue
damage after cerebral ischaemia. For these reasons, there
are serious doubts whether selective nNOS inhibitors are
applicable in acute stroke in humans.
Inhibition of inducible nitric oxide synthase:
aminoguanidine
In many respects, iNOS seems to be the most appropriate
target for pharmacological manipulation of nitric oxide
activity in acute cerebral ischaemia, as its appearance is
delayed for several hours after onset of ischaemia and its
eVects are protracted over several days, allowing time for
clinical intervention.16 Cerebral infarcts after permanent
MCAO are 28% smaller and motor deficits significantly
less in iNOS gene knockout mice compared with wild-type
mice.14 Furthermore, aminoguanidine, a relatively selective
iNOS inhibitor, shows time and dose dependent neuropro-
tective eVects—that is, the earlier the drug is given and the
higher the dose, the greater the reduction of infarct
volume. Reductions of 88% and 85% in infarct volume
have been reported when aminoguanidine is given 1 and 2
hours respectively after acute cerebral ischaemia in mice.21
Moreover, unlike other neuroprotective agents studied to
date, aminoguanidine still aVords significant neuroprotec-
tion even when administration is delayed by 24 hours after
onset of ischaemia, with reported infarct volume reduction
of 33%.22 The degree of reduction in infarct volume with
aminoguanidine correlates with the level of residual motor
function—that is, the neuroprotective eVect of aminogua-
nidine is functionally significant. Of interest, in one recent
study in rats the reduction of cerebral infarct volume was
not significantly less when aminoguanidine was given 24
hours after onset of ischaemia compared with starting
treatment at 12 hours.23
Importantly, aminoguanidine inhibits pathways other
than nitric oxide which may be important in mediating
cytotoxicity after acute cerebral ischaemia—that is, it is not
entirely selective for iNOS. For example, it also inhibits
polyamine oxidase in the brain. Polyamine oxidase
mediated release of 3-aminopropanal in high concentra-
tions after cerebral ischaemia is directly neurotoxic and
induces glial apoptotic cell death. After cerebral ischaemia,
there is a marked increase in polyamine oxidase activity
and aminoguanidine inhibits the consequent overproduc-
tion of 3-aminopropanal with associated reduction of cer-
ebral infarct volume.24 Through its inhibitory eVect on
advanced glycation end product formation, aminoguani-
dine is also known to be beneficial in preventing complica-
tions related to small vessel disease in diabetes mellitus
(retinopathy, neuropathy, and nephropathy) when given for
long periods—that is, months to years.25 However, this
property is of doubtful relevance to neuroprotection from
acute cerebral ischaemia.
The ability of aminoguanidine to disrupt a major delayed
neurodestructive pathway in acute cerebral ischaemia is an
important diVerence from the neuroprotective therapies so
3
far tried in humans. From the evidence of animal studies,
this property could be highly valuable in the treatment of
virtually all patients presenting with acute ischaemic stroke,
particularly those presenting to hospital outside the 6 hour
time window for most other neuroprotective drugs. Amino-
guanidine is already known to be safe and well tolerated in
humans25 and it may be given intravenously as well as orally.
Assuming that there is similar upregulation of brain iNOS in
humans as in rodents after acute cerebral ischaemia
(although this remains to be proved), aminoguanidine could
be a highly eVective neuroprotective drug. The therapeutic
potential of this and other selective iNOS inhibitors in acute
stroke should now be pursued with clinical trials.
DENIS O’MAHONY
Department of Geriatric Medicine, University of Birmingham, Clinical
Investigation Unit, Queen Elizabeth Hospital, Edgbaston, Birmingham
B15 2TH, UK
MARTIN J KENDALL
Department of Clinical Pharmacology, University of Birmingham,
Clinical Investigation Unit, Queen Elizabeth Hospital, Edgbaston,
Birmingham B15 2TH, UK
Dr O’Mahony, Department of Geriatric Medicine, University of Birmingham,
Clinical Investigation Unit, Queen Elizabeth Hospital, Edgbaston,
Birmingham B15 2TH, UK. email: d.omahony@bham.ac.uk
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 Downloaded from http://jnnp.bmj.com/ on July 8, 2017 - Published by group.bmj.com

Nitric oxide in acute ischaemic stroke: a target

for neuroprotection
DENIS O'MAHONY and MARTIN J KENDALL

J Neurol Neurosurg Psychiatry 1999 67: 1-3

doi: 10.1136/jnnp.67.1.1

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