L-Arginine-NO
pathway and CNS oxygen toxicity
NOEMI BITTERMAN1,4 AND HAIM BITTERMAN2,3,4
Naval Medical Institute, Medical Corps, Israeli Defense Forces, Haifa 31080;
2Department of Internal Medicine A, Carmel Medical Center, 3The Rappaport
Family Institute for Research in the Medical Sciences, and 4Faculty of Medicine,
Technion-Israel Institute of Technology, Haifa 34362, Israel
1Israel
central nervous system; hyperoxia; Nv-nitro-L-arginine methyl
ester; hypercapnia; nitric oxide; arginine; nitric oxide donors;
7-nitroindazole; S-nitroso-N-acetylpenicillamine
EXPOSURE TO OXYGEN at high pressure is commonly
employed in both military and professional (enriched
air) diving and in the clinical field of hyperbaric medicine (11, 19).
Breathing oxygen at high partial pressures produces
marked hemodynamic and neurological effects (8, 16).
Among the most significant are cerebral vasoconstriction (4, 16) and the hyperoxia-induced seizures, defined
and classified as generalized, tonic-clonic (grand mal)
seizures (16). At a certain point in time, an initial
oxygen-induced vasoconstriction is reported to be impaired and an increase in cerebral blood flow is observed (8, 16). It has been suggested (30) that this
increase in cerebral blood flow might be correlated with
the development of hyperoxia-induced seizures.
Exposure to hyperbaric oxygen (HBO) in combination with a low level of CO2, either from an external
source or because of an internal buildup of CO2, is a
common situation in underwater activity and certain
pathological conditions. An increased level of CO2 in the
breathing mixture leads to significant and dramatic
shortening of the latent period preceding seizures in
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both humans and experimental animals (16). Although
not fully proven, it is accepted that the increased
susceptibility to hyperoxia-induced seizures in the presence of hypercapnia is due to the cerebral vasodilator
effect of CO2 (8, 16).
The exact mechanism responsible for the hyperoxiainduced hemodynamic changes and neurological manifestations is still unknown. It is well accepted that
reactive oxygen species, which are produced in excess
on exposure to HBO and overwhelm the body’s normal
antioxidant defense systems, mediate the hyperoxic
insult (14, 29). However, the specific biological messengers responsible for the various expressions of the
hyperoxic insult have not yet been identified.
Nitric oxide (NO), a multifunctional small radical
molecule, is involved in a number of regulatory mechanisms (9, 21), including vasodilatation, neurotransmission and neuromodulation, inhibition of platelet aggregation, and modulation of leukocyte adhesion. On the
basis of the growing evidence for the biological importance of NO and the L-arginine-NO pathway in neural
and vasoregulatory mechanisms of the brain, we decided to investigate their role in the regulation of
central nervous system (CNS) oxygen toxicity.
To evaluate the role of NO in the pathophysiology of
hyperoxia-induced seizures, we selected some agents
affecting NO levels. On one hand, we used two inhibitors of NO synthase (NOS): Nv-nitro-L-arginine methyl
ester (L-NAME), a central and vascular NOS inhibitor,
and the novel 7-nitroindazole (7-NI), which is thought
to inhibit NOS solely in the brain in vivo (22). On the
other hand, we employed agents known to increase NO
levels: L-arginine (a precursor of NO), D-arginine (the
inactive D-isomer of L-arginine), and the NO donor
S-nitroso-N-acetylpenicillamine (SNAP). Two hyperoxic mixtures were used: pure HBO at 0.5 MPa [5
atmospheres absolute (ata)] and HBO with 5% CO2 at
the same pressure.
Our model was a freely moving rat with continuous
monitoring of the electroencephalogram (EEG). Animals were exposed to one of the two hyperoxic mixtures
after injection of different concentrations of the various
agents, until the onset of typical hyperoxia-induced
electrical discharges in the EEG.
MATERIALS AND METHODS
Animals. Experiments were carried out in male SpragueDawley rats, weighing 220–280 g and kept in a normal
day-night cycle. The rats were fed ad libitum on commercial
food and water.
Experimental procedures. Under equithensin anesthesia
[0.2–0.3 ml/100 g body weight ip; 1% chloral hydrate (wt/vol),
4% nembutal (wt/vol), and 1.3% MgSO4 (wt/vol) dissolved in
8750-7587/98 $5.00 Copyright r 1998 the American Physiological Society
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Bitterman, Noemi, and Haim Bitterman. L-Arginine-NO pathway and CNS oxygen toxicity. J. Appl. Physiol.
84(5): 1633–1638, 1998.—The involvement of the L-argininenitric oxide (NO) pathway in the pathogenesis of hyperoxiainduced seizures was studied by using agents controlling NO
levels. We selected two inhibitors of nitric oxide synthase, the
systemic inhibitor Nv-nitro-L-arginine methyl ester (L-NAME)
and the novel cerebral-specific inhibitor 7-nitroindazole, and
two generators of NO, the NO donor S-nitroso-N-acetylpenicillamine and the physiological precursor L-arginine. Rats with
chronic cortical electrodes were injected intraperitoneally
with different doses of one of the agents or their vehicles
before exposure to 0.5 MPa O2 and O2 with 5% CO2 at an
absolute pressure of 0.5 MPa. The duration of the latent
period until the onset of electrical discharges in the electroencephalogram was used as an index of central nervous system
O2 toxicity. The two nitric oxide synthase inhibitors L-NAME
and 7-nitroindazole significantly prolonged the latent period
to the onset of seizures on exposure to both hyperbaric O2 and
to the hypercapnic-hyperoxic mixture. Pretreatment with the
NO donor S-nitroso-N-acetylpenicillamine significantly shortened the latent period, whereas L-arginine, the physiological
precursor of NO, significantly prolonged the latent period to
onset of seizures. Our results suggest that the L-arginine-NO
pathway is involved in the pathophysiology of hyperoxiainduced seizures via various regulating mechanisms.
1634
L-ARGININE-NO
PATHWAY AND O2 TOXICITY
Fig. 1. Typical EEG from a rat exposed
to 0.5 MPa oxygen. A: air at atmospheric pressure (control). B: appearance of typical spike and wave electrical discharges at 0.5 MPa oxygen
(arrow). C: on return to atmospheric
pressure. Calibration: 1 s/50 V.
charges in the EEG, the chamber was immediately decompressed at a rate of 0.1 MPa/min. The experiment was
terminated after 50 min even if no discharges had been
recorded, to avoid complications because of pulmonary oxygen toxicity. In such a case, the duration of the latent period
was taken as 50 min.
Statistical methods. Latent period data (expressed in minutes) were transformed into a reciprocal form to enhance
normality (3) and are presented by a box-plot display showing
the extremes, the upper and lower quartiles (75 and 25
percentiles, respectively), and the medians. The transformed
mean values were compared by ANOVA, and Tukey’s test was
performed for comparison between specific pairs of data
means as a post hoc procedure. Two-group comparisons were
evaluated by Student’s t-test. Differences were considered
statistically significant when P , 0.05.
RESULTS
Figure 1 presents a typical EEG tracing from a rat
exposed to HBO: control recording made at atmospheric pressure (A), the appearance of typical spike
and wave electrical discharges in the EEG on exposure
to 0.5 MPa oxygen (B), and the EEG on return to
atmospheric pressure immediately after the onset of
seizures (C). There were no notable changes in the
baseline EEG, in the pattern of the seizures or their
severity, or in the behavior of the rats under the effect of
any of the agents employed in our study in any of the
experimental groups.
Figure 2 shows the reciprocal of the latent period to
the onset of seizures (the speed to seizure appearance)
after injection of various doses of L-NAME or 7-NI and
their vehicles (normal saline and peanut oil, respectively). A significant prolongation of the latency to the
onset of seizures (expressed by a decrease in the speed
to seizure appearance) was observed after intraperitoneal injection of each NOS inhibitor on exposure to
pure oxygen at 0.5 MPa. With L-NAME, the prolongation reached statistical significance compared with
vehicle at doses of 5 mg/kg or greater (P , 0.05 by
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absolute alcohol, propylene glycol, and water], the animal’s
skull was exposed and four stainless steel screws were
inserted into the bone bilaterally over the parietal and
posterior cortex, two screws at each hemisphere. Soldered
wires linked the screws to a miniature connector fixed to the
bone with self-cure acrylic. Rats recovered from surgery for 4
days before testing.
One hundred sixty-four rats were assigned at random to 18
groups with the following agents before exposure to 0.5 MPa
oxygen (n 5 9–10 rats/group): 1) L-NAME at a dose range of
1–20 mg/kg; 2) 7-NI (50 mg/kg); 3) SNAP at doses from
250–1,000 µg/kg; 4) L-arginine at doses of 30–300 mg/kg; 5)
D-arginine (300 mg/kg); 6) L-arginine (300 mg/kg) together
with L-NAME (20 mg/kg); and 7) D-arginine (300 mg/kg)
together with L-NAME (20 mg/kg). Additionally, 72 rats were
treated with L-NAME (1–20 mg/kg) or 7-NI (50 mg/kg; n 5 9
rats/group) before exposure to 0.5 MPa oxygen with 5% CO2.
All drugs (Sigma Chemical) were dissolved in saline, with the
exception of 7-NI, which was dissolved in peanut oil and sonicated. Both drugs and their appropriate vehicles were injected
intraperitoneally 10 min before the hyperbaric exposure.
A cable with a matching male plug was inserted into the
connector on the rat’s head so that a continuous two-channel
bipolar recording of the EEG from both hemispheres could be
obtained from the freely moving rat.
Each rat was exposed to hyperbaric mixtures only once in a
3-liter Plexiglas cage, placed inside a 150-liter hyperbaric
animal chamber (Technical Center for the Application of
Hyperbaric Oxygen-Roberto Galeazzi, La Spezia, Italy) that
was compressed by using air. Two hyperbaric mixtures were
used: pure oxygen and oxygen with 5% CO2, each at a
pressure of 0.5 MPa (5 ata). A constant flow of the gas mixture
of 2 l/min was employed to prevent the accumulation of CO2 in
the Plexiglas cage. The temperature of the chamber was
maintained at 22°C, with a transient rise in temperature of
up to 2°C during and shortly after compression, which was
carried out at a rate of 0.1 MPa/min.
The duration of the latent period, measured from the time
the desired pressure was reached until the appearance of
typical electrical discharges in the EEG, composed of highamplitude, fast activity with low-amplitude, slow activity
(spike and wave) (see Fig. 1), was used as the criterion for
CNS oxygen toxicity. On the appearance of electrical dis-
L-ARGININE-NO
PATHWAY AND O2 TOXICITY
Tukey’s test). 7-NI induced a marked prolongation of
the latent period (P , 0.01) at 50 mg/kg.
Figure 3 presents the effect of the two studied NOS
inhibitors on the onset of hyperoxia-induced seizures
on exposure to hypercapnic-hyperoxic mixture. In vehicle-treated rats breathing hyperoxic mixture with 5%
CO2 at 0.5 MPa, the duration of the latent period was
dramatically shorter compared with that on exposure
Fig. 3. Duration of latent period (1/time) to onset of electrical
discharges in EEG on exposure to 0.5 MPa oxygen with 5% CO2, after
injection of L-NAME or 7-NI or their vehicles (doses are in mg/kg ip).
Data are presented by box plots showing extremes, interquartile
range (25–75%), and medians. * P , 0.05 (Tukey’s test), ** P , 0.0001
(t-test): L-NAME or 7-NI pretreatment compared with control, respectively (n 5 9 rats/group).
to pure oxygen at the same pressure (5.7 6 0.3 vs.
19.1 6 4 min, respectively; P , 0.001 by t-test). As seen
in Fig. 3, pretreatment with L-NAME decreased the
speed to seizures (i.e., exerted a protective effect) in a
dose-related manner, reaching statistical significance
at a dose of 20 mg/kg (P , 0.05 by Tukey’s test). The
specific cerebral NOS inhibitor 7-NI caused a highly
significant, almost the maximal possible, prolongation
of the latent period (P , 0.0001 by t-test).
The effect of agents that increase NO levels and
their control on the latent period to HBO-induced
seizures is presented in Figs. 4 and 5. Pretreatment
with L-arginine, the natural physiological precursor of
NO, postponed HBO toxicity, as presented by a doserelated decrease in the speed of onset of hyperoxicinduced seizures, reaching statistical significance at a
dose of 300 mg/kg (P , 0.05 by Tukey’s test). The
inactive isomer D-arginine (300 mg/kg), given as a
control, had no effect on the latent period to HBOinduced seizures (Fig. 4). In contrast, the NO donor
SNAP, given before HBO exposure, caused a significant
dose-related decrease in the latent period, reaching a
significant enhancement of toxicity at the dose of 1,000
µg/kg (P , 0.05) (Fig. 5).
Combined treatment with either L-arginine or
D-arginine (300 mg/kg) given 10 min after L-NAME (20
mg/kg) provided a significant protection (P , 0.05 by
Tukey’s test) against hyperoxia-induced seizures compared with control. However, the combined effect of the
the two protective drugs on the latent period was not
significantly different from the protection achieved
with L-NAME alone.
Figure 6 summarizes the relative effect of each
NO-controlling agent on the mean latency period to
seizures normalized against its respective control. As
can be seen, all agents tested herein increased the time
Fig. 4. Duration of latent period (1/time) to onset of electrical
discharges in EEG, on exposure to 0.5 MPa oxygen, after injection
with L-arginine or D-arginine (doses are in mg/kg ip). Data are
presented by box plots showing extremes, interquartile range (25–
75%), and medians. * P , 0.05 (Tukey’s test) compared with control
(n 5 9 rats/group).
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Fig. 2. Duration of latent period (1/time) to onset of electrical
discharges in EEG on exposure to 0.5 MPa oxygen after injection with
Nv-nitro-L-arginine methyl ester (L-NAME) or 7-nitroindazole (7-NI)
and their vehicles (doses are in mg/kg ip). Data are presented by box
plots showing extremes, interquartile range (25–75%), and medians.
* P , 0.05 (Tukey’s test), ** P , 0.01 (t-test): L-NAME or 7-NI
pretreatment compared with control, respectively (n 5 9 rats/group).
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L-ARGININE-NO
PATHWAY AND O2 TOXICITY
to onset of seizures, except for SNAP, which shortened
the latent period.
DISCUSSION
In the present study, we evaluated the involvement of
the L-arginine-NO pathway in the pathogenesis of
hyperoxia-induced seizures by using various agents
controlling NO levels. It has previously been suggested
by Oury et al. (25) and Zhang et al. (33) that NO is an
important mediator of the pathophysiology of oxygen
toxicity by using Nv-nitro-L-arginine as a NOS inhibitor. Both studies utilized mortality and the appearance
of clinical convulsions as indexes of oxygen toxicity. We
Fig. 6. Normalized data for duration to onset of seizures expressed in
percent relative to control vehicle group for each treatment.
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Fig. 5. Duration of latent period (1/time) to onset of electrical
discharges in EEG on exposure to 0.5 MPa oxygen, after injection of
S-nitroso-N-acetylpenicillamine (SNAP; doses are in µg/kg ip). Data
are presented by box plots showing extremes, interquartile range
(25–75%), and medians. * P , 0.05 (Tukey’s test) compared with
control (n 5 9 rats/group).
extended the previous observations by using the objective, well-defined parameter of typical electrical discharges in the EEG as a criterion for CNS oxygen
toxicity. We also evaluated the role of NO on exposure to
hypercapnic-hyperoxic mixture. To further evaluate
the role of NO, we selected agents controlling NO levels
in both directions: two NOS inhibitors, the commonly
used systemic NOS inhibitor L-NAME and the novel
cerebral NOS inhibitor 7-NI, as well as the two NO
generators, the NO donor SNAP and the physiological
precursor L-arginine.
L-NAME and 7-NI, two NOS inhibitors, postponed
the appearance of hyperoxic seizures significantly, without any noticeable side effects on the EEG or on the
animals’ behavior.
There are a number of possible explanations for the
protective effects of NOS inhibitors against hyperoxiainduced seizures that are related to the vasodilatory,
prooxidative, and neuromodulatory functions of NO.
Exposure to HBO is accompanied by cerebral vasoconstriction (4, 16), which is subsequently followed by
cerebral vasodilatation. It has been suggested that this
breakdown of cerebral vasoregulation and the resultant increase in cerebral blood flow correlate with the
development of hyperoxia-induced seizures (30). The
basic mechanisms of the vasoregulatory effects of oxygen are not fully understood, and it has been suggested
that NO, which is known to mediate cerebral vasodilatation (13), may be involved. On the basis of this, the
inhibition of NO production by pretreatment with NOS
inhibitors such as L-NAME or 7-NI may postpone the
breakdown of hyperoxic cerebral vasoconstriction, thus
attenuating the dramatic preseizure increase in cerebral blood flow and the inflow of oxygen and reactive
oxygen intermediates to the brain. It may therefore be
suggested that the NOS inhibitors postponed HBOinduced seizures by diminishing cerebral vasodilatation.
The protective effect of both NOS inhibitors was also
demonstrated in our study on exposure to a hyperoxichypercapnic mixture (oxygen with 5% CO2 at a pressure of 0.5 MPa). As already known in human and
animal models (8, 16), hypercarbia, which is common in
diving and in various pathological clinical conditions,
augments the neurotoxic effects of HBO by shortening
the duration of the latent period to the onset of seizures
and decreasing the threshold toxic pressure of oxygen.
It is accepted that this increased toxicity on exposure to
elevated concentrations of CO2 is caused by cerebral
vasodilatation (8, 16), thus increasing the inflow of
reactive oxygen species or other humoral toxic mediators to the brain. In various studies NO has been
implicated in the mediation of hypercapnic cerebral
vasodilatation (5, 12), and NOS inhibitors were reported to reduce the increase in cerebral blood flow
elicited by hypercapnic challenges (5, 27). We therefore
suggest that the protective effect of NOS inhibitors
given before exposure to HBO with 5% CO2 might also
be explained by decreased hypercapnia-induced NO
production, reducing cerebral vasodilatation.
L-ARGININE-NO
PATHWAY AND O2 TOXICITY
pathway in hyperoxia-induced seizures. We selected
the NO donor SNAP and L-arginine, which is the
physiological precursor for NO production. On the basis
of our experiments with NOS inhibitors, we assumed
that increasing tissue levels of NO by pretreatment
with either L-arginine or the NO donor would enhance
the development of HBO-induced toxicity because of
the vasodilatory, prooxidant, and neuromodulatory activity of NO. Pretreatment with the NO donor SNAP
indeed confirmed our expectations. The significant
NO-induced decrease in the duration of the latent
period to the onset of seizures is expressed in Fig. 5 by
an increase in the speed of development of seizures. In
contrast, pretreatment with L-arginine induced a doserelated protective effect against hyperoxic seizures.
D-arginine, the inactive isomer of L-arginine, failed to
induce any significant change in the latent period to
convulsions, both when given alone and in combination
with L-NAME. The rather unexpected specific effect of
L-arginine can be explained by both NO-dependent and
NO-independent mechanisms (10).
It is well known that NO exerts opposing effects,
depending on the tissue redox state (18), on differences
between species and on the concentrations and the
source of NO.
By using L-NAME we probably inhibited the vasodilatory, epileptogenic, and prooxidative effects of NO. It is
suggested that by giving L-arginine we may have
uncovered the opposing effects of NO, namely, its
antiepileptic (6), antioxidative (15), and cytoprotective
(32) manifestations.
Another possibility that cannot be excluded is that
L-arginine protected against hyperoxia-induced seizures via routes that are unrelated to the production
of NO. In addition to the NOS pathway, L-arginine is
also metabolized to L-ornithine and urea by arginase.
L-Ornithine is further metabolized to polyamines (spermine, putrescine, spermidine) and L-proline. There are
reports suggesting possible involvement of L-arginine
and polyamines in the pathophysiology of HBO toxicity
(7, 20). In this regard, it has been demonstrated that
polyamines inhibit the oxidation and peroxidation of
unsaturated fatty acids both in vivo and in vitro, and it
is assumed that their role in cellular defense mechanisms against oxygen toxicity is that of oxygen-reactive
species scavengers (7). The differences between the
effects of SNAP and L-arginine support the hypothesis
that at least part of the effect of L-arginine might be
mediated through protective mechanisms unrelated
to NO.
Because in our study both L-NAME and L-arginine
given alone protected rats against hyperoxia-induced
seizures, it is not surprising that given together they
had no opposing effects in the prevention of CNS
oxygen toxicity. Another study in which the protective
effect of L-NAME was not reversed by addition of
L-arginine has been reported by Shimizu et al. (28) in
an oxidative model of H2O2-induced endothelial cell
injury.
Altogether, our findings can be explained by the
ambiguous and dual face exhibited by NO, which acts
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As seen in Fig. 6, there are considerable differences
in the relative effectiveness of the two NOS inhibitors
studied herein in protection against hyperoxia alone
and against exposure to breathing a hyperoxic-hypercapnic mixture. L-NAME was more effective on exposure to pure HBO than on exposure to the hypercapnichyperoxic mixture (a 123% increase in the duration of
the latent period to the onset of seizures compared with
49%, respectively). In contrast, 7-NI, a selective cerebral NOS inhibitor, was much more effective in hypercapnic-hyperoxic exposure than in pure oxygen (223%
increase in the latent period vs. 71%, respectively). The
cerebral NOS inhibitor 7-NI produced almost complete
protection (P , 0.0001 compared with its vehicle)
against the hypercapnic mixture, which is the best
neuroprotection we have ever achieved with any compound tested against HBO seizures.
The dramatic protective effect of the cerebral NOS
inhibitor in hyperoxic-hypercapnic exposure, as well as
its superior protective effect compared with the systemic NOS inhibitor L-NAME, suggests that CO2 enhances CNS oxygen toxicity not only by inducing
vasodilatation but also via direct neural mechanisms
causing an increased sensitivity to HBO.
The second explanation for the protective effect of
NOS inhibitors may be related to the prooxidative
actions of NO. It is well accepted that exposure to HBO
increases the generation of reactive oxygen species
(superoxide and hydroxyl radicals, hydrogen peroxide,
and so on) (14, 29). Under these conditions of increased
tissue levels of oxygen free radicals, NO may augment
cytotoxicity. NO and superoxide radical react rapidly to
form peroxynitrite anion (2). Peroxynitrite is directly
cytotoxic and also decomposes into other toxic species
in the tissues (31). Recent studies suggest that CO2
modulates most of the reactions of peroxynitrite in
biological systems (26), acting as a true catalyst of the
decomposition of peroxynitrite, a finding that can explain the increased sensitivity to HBO on exposure to
hypercapnic-hyperoxic mixtures. It has also been shown
that NO reacts with hydrogen peroxide to produce
singlet oxygen, a highly reactive and toxic form of
oxygen radical (23). Hence, treatment with NOS inhibitors such as L-NAME and 7-NI, leading to a decrease in
tissue NO levels, may attenuate the generation of
cytotoxic intermediates formed by the interaction of
NO with oxygen-reactive species. We also cannot exclude direct antioxidant effects of NOS inhibitors,
unrelated to NO production, such as have recently been
shown with L-NAME, which reduce endothelial cell
injury induced by hydrogen peroxide (28).
A third mechanism by which NOS inhibitors protect
against HBO-induced seizures may be related to the
neuromodulatory effects of NO, such as increasing the
release of the potentiating neurotransmitters glutamate and aspartate (17). These effects of NO might
explain the antiepileptic effect of L-NAME in other
experimental models of epilepsy, such as pentyenetetrazol-induced seizures (24).
Increasing tissue NO levels is an alternative approach that can enlighten the role of the NO-L-arginine
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L-ARGININE-NO
PATHWAY AND O2 TOXICITY
both as a cytotoxic and a cytoprotective agent. A variety
of direct and indirect cytotoxic and cytoprotective effects of NO have been reported in different models of
cellular injuries (1, 10). Specific redox-based neuroprotective and neurodestructive effects of NO were reported by Lipton et al. (18). Furthermore, NO has also
been shown to specifically exert cytoprotective effects in
oxidative stress (32). The balance between the cytotoxic
and cytoprotective effects of NO, and the net outcome of
these effects in any given pathophysiological condition,
seems to depend on myriad factors and mechanisms
that are presently only partly elucidated in the specific
case of CNS oxygen toxicity. Moreover, it is possible
that the mechanisms underlying the protection against
HBO-induced seizures may be partly NO dependent
and partly NO independent, acting through other
mechanisms. Our results support a role for the
L-arginine-NO pathway in the pathophysiology of hyperoxia-induced seizures.
Received 31 May 1997; accepted in final form 10 December 1997.
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We thank Richard Lincoln for help in the preparation of the
manuscript. The technical assistance of Geula Diamant is greatly
appreciated.
This study was carried out in adherence with the National
Institutes of Health guidelines for the use of experimental animals.
The opinions and assertions presented are those of the authors and
are not to be construed as the official views of the Israel Naval
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Address for reprint requests: N. Bitterman, Israel Naval Medical
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