brain research 1574 (2014) 77–83

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Research Report

A comparison of factors involved in the development

of central nervous system and pulmonary oxygen
toxicity in the rat

Mirit Eynann, Nitzan Krinsky, Adi Biram, Yehuda Arieli, Ran Arieli
Israel Naval Medical Institute, Israel Defense Forces Medical Corps, Box 22, Rambam Health Care Campus,
P.O. Box 9602, 3109601 Haifa, Israel

art i cle i nfo ab st rac t

Article history: Central nervous system oxygen toxicity (CNS-OT) can occur in humans at pressures above
Accepted 30 May 2014 2 atmospheres absolute (ATA), and above 4.5 ATA in the rat. Pulmonary oxygen toxicity
Available online 11 June 2014 appears at pressures above 0.5 ATA. We hypothesized that exposure to mild HBO following

Keywords: extreme exposure might provide protection against CNS, but not pulmonary oxygen toxicity.

CNS-oxygen toxicity We measured the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione

Antioxidant enzymes peroxidase (GPX), and nitrotyrosine and nNOS levels in the brain and lung in the following

Hyperbaric oxygen groups: (1) Sham rats, no pressure exposure (SHAM); (2) Exposure to 6 ATA oxygen for 60% of
latency to CNS-OT (60%LT); (3) Exposure to 6 ATA for 60% of latency to CNS-OT, followed by
20 min at 2.5 ATA for recovery (REC); (4) Exposure to 6 ATA for 60% of latency to CNS-OT,
followed by 20 min at 2.5 ATA oxygen and a subsequent increase in pressure to 6 ATA until the
appearance of convulsions (CONV); (5) Control rats exposed to 6 ATA until the appearance of
convulsions (C). SOD and CAT activity were reduced in both brain and lung in the REC group.
GPX activity was reduced in the hippocampus in the REC group, but not in the cortex or the
lung. nNOS levels were reduced in the hippocampus in the REC group. Contrary to our
hypothesis, no difference was observed between the brain and the lung for the factors
investigated. We suggest that at 2.5 ATA and above, CNS and pulmonary oxygen toxicity
may share similar mechanisms.
& 2014 Elsevier B.V. All rights reserved.

1. Introduction sometimes without any warning symptoms. CNS-OT can

occur in humans at pressures above 2 atmospheres absolute
Central nervous system oxygen toxicity (CNS-OT) represents a (ATA) (Hampson and Atik, 2003; Yildiz et al., 2004), and above
major risk for combat divers. It is characterized by convulsions 4 ATA in the rat (Arieli et al., 2001; Pilla et al., 2013). Pulmonary
similar to epileptic seizures and sudden loss of consciousness, oxygen toxicity appears in humans at pressures above 0.5 ATA

n
Corresponding author. Fax: þ972 4 8693258.
E-mail addresses: estie@mail.idf.il, emirit@netvision.net.il (M. Eynan), k_nitz@walla.com (N. Krinsky), adi.biram@gmail.com (A. Biram),
yarieli@netvision.net.il (Y. Arieli), rarieli@netvision.net.il (R. Arieli).

http://dx.doi.org/10.1016/j.brainres.2014.05.051
0006-8993/& 2014 Elsevier B.V. All rights reserved.
78 brain research 1574 (2014) 77–83
(Jackson, 1985) and in rats above 1 ATA (Harabin et al., 1990).
One difference between these two toxicities is the latency to
their appearance. Whereas CNS-OT can appear within min-
utes, the development of pulmonary oxygen toxicity takes
several hours, depending on the ambient pressure.
In a study that screened over 2500 dives with closed-
circuit apparatus, Arieli et al. (2006) found that symptoms
related to CNS-OT were reported during dives to 3–6 m sea
water, but not at 2 m. They suggested that in man, recovery
from CNS-OT may take place at a level of hyperoxia where
there is a continued risk of pulmonary oxygen toxicity. Arieli
et al. (2002) and Arieli (2003) proposed an algorithm to
calculate the risk of CNS-OT while diving with closed-circuit
oxygen rebreathers. This algorithm was calculated using a rat
model, based on the increase in the cumulative oxygen load
index at a PO2 in excess of the threshold for steady state
production and removal of reactive oxygen species (ROS)
(Arieli and Hershko, 1994). The underlying assumption was
that recovery of the factors leading to the development of
CNS-OT will take place during the steady state. To validate
this assumption, Arieli et al. (2008) investigated whether
recovery from CNS-OT would take place at a PO2 higher than
normoxia. It was found that recovery from CNS-OT in rats
takes place at a PO2 between 0.21 and 3 ATA. This later
investigation was conducted on a rat model, and was defined
as a pioneer study with a view to establishing the hypothesis.
ROS and reactive nitrogen species (RNS) such as peroxyni-
trite, produced by the reaction of NO with superoxide, are
both factors that play a major role in the generation of
oxygen toxicity (Bitterman and Bitterman, 1998; Bitterman
et al., 1994; Demchenko and Piantadosi, 2006; Elayan et al.,
2000; Ohtsuki et al., 1992; Oury et al., 1992; Torbati et al.,
1992). Living cells have developed a number of mechanisms
for coping with continuous exposure to ROS, among others
antioxidant enzymes and low molecular weight antioxidants
such as vitamin C, vitamin E and glutathione, which sca-
venge the ROS. The antioxidant enzymes include three types
of superoxide dismutase (SOD), which transform the super-
oxide ion into hydrogen peroxide (H2O2). Catalase and glu-
tathione peroxidase catalyze H2O2 to O2 and H2O. Under
normal conditions, there is a state of equilibrium between
ROS generation and ROS scavenging. In oxidative stress, how-
ever, this equilibrium is disturbed, and an increase in the
level of ROS may result in cell injury (Ames et al., 1993;
Davies, 1987; Halliwell, 1992).
The cumulative oxygen load index may not be the same
in the brain and the lung at different oxygen pressures.
Demchenko et al. (2007) demonstrated in a rat model that
at 2–2.5 ATA and above pulmonary injury is largely due to a
non-inflammatory process, whereas at lower pressures it is
due to a direct inflammatory process. In addition, at hyper-
baric pressure a neurogenic CNS affects the development of
pulmonary oxygen toxicity. This phenomenon was initially
described by Bean and Rottschafer (1938), Bean and Smith
(1953), and Bean and Johnson (1955); Demchenko et al. (2011,
2012) continue to search for its specific underlying mechan-
ism. It should be pointed out that no neurogenic component
has of yet been reported as part of the role played by the
sympathetic nervous system in the generation of pulmonary
oxygen toxicity in humans (Winklewski et al., 2013).
We suggest it may be possible to use the finding that
CNS-OT recovers in a rat model at a PO2 of 2–3 ATA to
differentiate between the processes leading to CNS and
pulmonary oxygen toxicity. We hypothesized that after
exposure to 6 ATA, even without reaching the point of
convulsion, a reduction of the HBO pressure to 2.5 ATA will
bring about recovery of the CNS-OT, whereas the inflamma-
tory processes leading to pulmonary oxygen toxicity may
continue to develop.
2. Results
2.1. Antioxidant enzyme activity
Total SOD activity was significantly reduced in the brain (pre-
dominantly in the hippocampus, po0.01) and the lung (po0.05)
in the REC group, compared with the other groups (Fig. 2). The
same pattern was obtained in the brain for catalase (po0.05),
while in the lung, catalase activity was reduced in the REC group
compared with the 60%LT group and with the CONV and C
groups (po0.05, Fig. 3). There was no significant difference in the
activity of SOD and catalase between the CONV and C groups.
GPX activity was reduced in the hippocampus in the REC group
compared with the 60%LT group (po0.01), but did not change in
the cortex. In the lung, GPX activity increased in the REC and
CONV groups compared with 60%LT group (Fig. 4).
2.2. Nitrotyrosine and nNOS immunoblotting
Using Western blot technique, we found differences between
the groups for proteins labeled by an antibody specific to
nitrotyrosine. In the cortex and the hippocampus we
observed nitrotyrosylation of proteins at 65 kDa and 75 kDa
(Fig. 5a), whereas in the lung nitrotyrosylation was observed
only at 65 kDa (Fig. 5b). NT levels at 75 kDa in the cortex were
elevated in the REC group compared with the other groups
(po0.01, Fig. 6). No changes in NT at 75 kDa levels were
observed in the hippocampus. No significant change was
observed in the cortex or the hippocampus at 65 kDa. NT
levels at 65 kDa in the lung were reduced in the REC group
compared with the C group (po0.05, Fig. 7).
nNOS levels were significantly reduced in the hippo-
campus in the REC group compared with the SHAM
General scheme of the
experimental protocol
1 ATA
Air Oxygen Oxygen
Pressure (ATA)
2.5 ATA
20 min 20 min
60% of Recovery Time until
latency time convulsions
6 ATA
Time (min)
Fig. 1 – General scheme of the experimental protocol.
 brain research 1574 (2014) 77–83 79

Lung
250kDa
0.30 Cortex
150 kDa
0.25 Hippocampus
100 kDa
(U/µg protein)
SOD activity

0.20
75kDa
0.15

0.10 * *
50 kDa
0.05 * 37 kDa
0.00

Fig. 2 – SOD activity in the lung, cortex and hippocampus in

250kDa
the five experimental groups. Results are expressed as
mean7SD. (*) po0.01 vs. 60%LT and C groups. 150 kDa
100 kDa
75kDa
60
(nmol/min/µg protein)
Catalase activity

50 Cortex
50 kDa
Hippocampus
40
37 kDa
30
*
20 Fig. 5 – (a) Representative Western blot of homogenates from
*
10 the brain stained with polyclonal antibody reactive to
0 nitrotyrosine. (b) from the lung. Two major molecular weight
bands (75 kDa and approximately 65 kDa) were found in the
brain, whereas in the lung one major molecular weight band
(65 kDa) was found.
(nmol/min/µg protein)

800 *
*
Catalase activity

Lung
SHAM 60%LT REC CONV C
600 *
NT (CRX) 75 kDa
400
Actin
200
normalized to actin ( % of sham)

0
75 kDa protein nitration level

Cortex
180
150
* Hippocampus

Fig. 3 – (a) CAT activity in the cortex and hippocampus in the 120
five experimental groups. (*) po0.05 vs SHAM, 60%LT and C 90
60
groups. (b) CAT activity in the lung in the five experimental
30
groups. Results are expressed as mean7SD. (*) po0.05. 0

Fig. 6 – Nitrotyrosine levels in the cortex and hippocampus

Lung
in the five experimental groups at 75 kDa (n ¼ 5).
Cortex
(nmol/min/µg protein)

Representative bands are shown from the cortex.

5.0 Hippocampus Nitrotyrosine levels were related to actin. Nitrotyrosine
GPX activity

4.0 levels in the cortex were elevated in the REC group

3.0 compared with the other groups. (*) po0.01. Data are
2.0 expressed as mean7SD.
*
1.0
*
0.0
and 60%LT groups (po0.01 and po0.05, respectively),
increasing slightly (without reaching statistical signifi-
cance) in the CONV group (Fig. 8). No change was
Fig. 4 – GPX activity in the cortex, hippocampus and lung in found in levels of nNOS in the cortex (not shown). In
three experimental groups. Results are expressed as the lung, levels of nNOS were too low for bands to be
mean7SD. (*) po0.01 vs. the 60%LT group. observed.
80 brain research 1574 (2014) 77–83

SHAM 60%LT REC CONV C activity in the brain (mainly the hippocampus), following
NT (Lung) 65 kDa
reduction of the oxygen pressure from 6 ATA (the pressure at
Actin which rats reach the point of convulsion) to 2.5 ATA for a period
of 20 min, the time needed for completion of the recovery phase
Lung
(Arieli et al., 2008). When the pressure was again raised to 6 ATA
normalized to actin (% of sham)
65 kDa protein nitration level

Cortex
180
Hippocampus
until convulsions appeared (CONV group), the activity of these
150 enzymes increased, reaching the same levels that had been
120 * present at 6 ATA before the recovery phase. The reduction in
90 their activity at the end of the recovery phase, seen predomi-
60 nantly in the hippocampus, corroborates the findings of Arieli
30 et al. (2008) that recovery from central nervous system oxygen
0 toxicity in the rat takes place at oxygen pressures between 1
and 3 ATA. The pattern of activity of SOD and catalase with the
change in pressure was similar in the brain and the lung. This
Fig. 7 – Nitrotyrosine levels in the lung in the five
suggests that their activity is related to oxidative stress, and
experimental groups at 65 kDa (n ¼ 5). Representative bands
that they are not specific to the development of CNS-OT. The
are shown from the lung. (*) po0.01 vs. 60%LT, CONV and C
only difference we found between the brain and the lung was in
groups. Data are expressed as mean7SD.
the activity of GPX, which was still elevated in the lung in the
REC group, whereas in the hippocampus it was reduced after
the recovery phase. This may indicate that GPX is not involved
PC SHAM 60%LT REC CONV C
in the development of CNS-OT in the same way as it is in
nNOS pulmonary oxygen toxicity.
CNS and pulmonary oxygen toxicity cannot be considered
Actin
two distinct and separate entities on exposure to HBO at
pressures above 2.5 ATA. At these pressures, CNS-OT does have
an effect on pulmonary oxygen toxicity, based on the assump-
0.7 tion that the dosage of oxygen that induces CNS-OT will also
0.6 activate the sympathetic nervous system (Bean and Johnson,
nNOS/actin pixel ratio

1955; Bean and Rottschafer, 1938; Bean and Smith, 1953).

0.5
Demchenko et al. (2007, 2011, 2012) demonstrated linkage
0.4 between the generation of CNS and pulmonary oxygen toxicity
0.3 after exposure to 5 or 6 ATA. A massive sympathetic outflow
mediated by the CNS depressed left ventricular function, leading
0.2
to the sudden development of cardiogenic pulmonary edema.
*
0.1 Moreover NO, produced by nNOS in the periventricular regions
0 of the brain, plays a crucial role in the events leading up to CNS-
OT and the associated sympathetic outflow that brings about the
pulmonary damage. At 2 ATA and above, pulmonary injury is
Fig. 8 – nNOS levels in the hippocampus in the five largely due to a non-inflammatory process, unlike at lower
experimental groups. Results are shown as representative pressures, when it is due to a direct inflammatory process.
bands produced by Western blot analysis. nNOS levels were The results obtained in the present study, namely a reduction
found to be significantly lower in the REC group compared of SOD and catalase activity and a decrease in the level of nNOS
with the SHAM and 60%LT groups. (*) po0.01 and 0.05, following the recovery phase, provide support for this linkage.
respectively. Data are expressed as mean7SD. Our results imply that reduction of the HBO pressure from 6 to
2.5 ATA leads to recovery of both CNS and pulmonary oxygen
toxicity. However, all of these findings were obtained from a
small animal model, not in humans exposed to high pressure O2.
A measure of uncertainty remains as to whether there may be
3. Discussion linkage between the generation of CNS and pulmonary oxygen
toxicity via the sympathetic nervous system in larger animal
In the present study, we investigated the hypothesis that models or in humans (Winklewski et al., 2013). This lack of
exposure of rats to conditions in which both CNS and pulmon- information may cast doubt on the implications of our results for
ary oxygen toxicity develop (6 ATA O2), and a subsequent divers who use closed-circuit oxygen apparatus.
reduction of pressure to 2.5 ATA, would result in recovery of There is evidence that SOD regulates NO availability for
the factors involved in the generation of CNS-OT, whereas vasodilation during HBO (Chavko et al., 2003; Demchenko et al.,
levels of the same factors in the lung which are involved in 2002; Elayan et al., 2000; Oury et al., 1992). A high level of SOD
pulmonary oxygen toxicity would increase. To test the hypoth- (by administration or in overexpressed transgenic mice) sca-
esis, we measured the activity of the antioxidant enzymes SOD, venges O2 . Because there is then less superoxide available to
catalase and GPX in the brain (cortex and hippocampus) and in engage with NO and produce the molecule peroxynitrite, the
the lung. We found a reduction in SOD, catalase and GPX large amount of NO that is left will augment cerebral blood flow
 brain research 1574 (2014) 77–83
(CBF) in the brain, with a resultant increase in oxygenation and
hence in susceptibility to CNS-OT (Demchenko et al., 2002; Liu
et al., 2012; Oury et al., 1992). In the present study, the activity of
SOD and the level of nNOS were reduced in the brain at the end
of recovery phase, but increased (mainly in the hippocampus)
when convulsions appeared. A smaller amount of SOD may
leave higher levels of superoxide to react with NO to generate
OONO  . Demchenko et al. (2002) demonstrated that regional
CBF increased throughout 60 min of exposure to HBO at 5 ATA
in mice with overexpressed SOD3 (SODþ/þ3). This was asso-
ciated with earlier onset of EEG spikes, in comparison with wild
type and SOD  / 3 mutant mice. The authors provide clear-cut
evidence that SOD3 regulates CBF by controlling the equilibrium
between NO and O2 . An increase in CBF via NO overproduction
is known to be one of the mechanisms leading to the onset of
CNS-OT (Chavko and McCarron, 2006; Demchenko et al., 2000,
2001). Finally, low activity of SOD will result in lower levels of
NO, because the latter is engaged by the superoxide to generate
OONO  .
Peroxynitrite, formed by the interaction of superoxide and
nitric oxide, is a strongly reactive oxidant. It was shown to
interact with proteins and cause nitrosative modification of
tyrosine residues to form nitrotyrosine. Nitrotyrosine, a marker
of OONO  , increased in the present study at the end of the
recovery phase in the cortex, but not in the hippocampus. In the
recovery phase, the possible reduction in SOD and NO may be a
factor that provides protection against CNS-OT. Previous studies
suggested that the cortex is less dominant in the development
of CNS-OT than the hippocampus and the striatum (Wang et al.,
1998). Higher levels of peroxynitrite in the cortex, together with
lower levels of SOD activity at the end of the recovery phase,
may indicate less free NO. This may have the beneficial effect of
inducing a recovery process from oxygen toxicity when the HBO
pressure is reduced from 6 to 2.5 ATA.
Results for antioxidant enzyme activity following HBO expo-
sure in studies using the rat as an animal model are contra-
dictory. In some of these studies, rats were exposed to pressures
employed in therapeutic HBO regimens (2.5–3 ATA). Harabin
et al. (1990) demonstrated no change in SOD, catalase or GPX
activity following exposure to 2.8 ATA for 4 h. Matsunami et al.
(2010) demonstrated a reduction in SOD activity following
exposure to 2.8 ATA for 2 h. However, in the study by Oter
et al. (2005), the activity of SOD increased as the pressure
increased from 2, through 2.5 and 3 ATA for 2 h, which indicates
oxidative stress. In the study by Korkmaz et al. (2008), SOD
activity increased in relation to exposure time at 3 ATA, and
GPX activity was inconsistent throughout the exposure time. In
studies that used higher pressures (5–6 ATA) which induced
convulsions, SOD, catalase and GPX were reduced in the cortex
and hippocampus compared with controls following exposure
to 6 ATA for 20 min or until convulsions (Liu et al., 2012; Zhang
and Piantadosi, 1991). Despite these contradictory results, we
suggest that the reduction in the activity of these enzymes after
pressure was lowered to 2.5 ATA might reflect a decrease in
oxidative stress.
In summary, our results may suggest that reduction of the
HBO pressure from 6 to 2.5 ATA leads to recovery from both
CNS and pulmonary oxygen toxicity in a rat model. With the
exception of GPX, there was no clear-cut difference in the
pattern of any of the investigated factors for the brain and the
81
lung throughout the different phases of exposure to HBO. We
therefore suggest that at pressures of 2.5 ATA and above,
CNS-OT and pulmonary oxygen toxicity probably share simi-
lar mechanisms. However, GPX may be regarded as a factor
more directly involved in the development of CNS-OT. The
method employed in the present investigation might be used
in further studies in the search for additional factors involved
specifically in CNS-OT.
4. Experimental procedure
4.1. Animals and maintenance
Forty male Sprague-Dawley rats weighing 250–300 g were
housed in plastic cages under standard conditions, with free
access to drinking water and standard chow. They were kept in
an 8-h light:16-h dark cycle, and the ambient temperature was
maintained at 24 1C. The Animal Care Committee of the Israel
Ministry of Defense approved the experimental procedure, and
the rats were handled and surgical procedures performed in
accordance with internationally accepted humane standards.
4.2. Protocol
Fig. 1 shows a general scheme of the experimental protocol.
Rats were divided randomly into five groups. (1) Sham rats,
placed in the hyperbaric chamber breathing room air but not
exposed to pressure, to mimic the stress of being put in the
chamber (SHAM, n¼6). (2) Exposure to 6 ATA for 60% of latency
to CNS-OT (60%LT, n¼ 8). (3) Exposure to 6 ATA for 60% of
latency to CNS-OT, followed by 20 min exposure to 2.5 ATA
oxygen for recovery from CNS-OT (REC, n¼8). (4) Exposure to 6
ATA for 60% of latency to CNS-OT, followed by 20 min exposure
to 2.5 ATA oxygen and a subsequent increase in pressure to
6 ATA until the appearance of convulsions (CONV, n¼8); (5)
Control rats exposed to 6 ATA until the appearance of convul-
sions (C, n¼6). After each exposure, tissue from the lung, cortex
and hippocampus was quickly removed, placed in liquid nitro-
gen, and kept at  80 1C until analysis.
Before commencing the experiment, each of the rats in
groups 2–5 was exposed twice to 6 ATA oxygen in order to
determine its latency to CNS-OT. There was an interval of one
week between consecutive exposures to ensure there would
be no effect on latency from one exposure to the next (Arieli
and Hershko, 1994). Latency to CNS-OT did not differ by more
than 15% between these exposures.
4.3. Exposure to HBO
The rat was put in the experimental cage, which was placed
inside a 150-l hyperbaric chamber (Roberto Galeazzi, La
Spezia, Italy). The flow of gas through the cage was controlled
by two needle valves. A small portion of the outgoing gas was
directed out of the pressure chamber (controlled by another
needle-valve), passed through a flowmeter, and was sampled
by an oxygen analyzer (Servomex Model 571, Crowborough,
East Sussex, UK) which monitored the concentration of O2 in
the experimental cage. The temperature in the cage was
82 brain research 1574 (2014) 77–83
maintained within a thermoneutral range (2771 1C) to avoid
any effect of temperature on CNS-OT. When the desired
pressure was reached (6 ATA), a period of 20 min was allowed
for acclimation to the experimental conditions, during which
air flowed through the cage at approximately 5 l/min. At the
end of this period, the flow of air was immediately replaced by
pure oxygen at a fast flow rate of 15 l/min for 1 min to replace
the cage's atmosphere. When the oxygen level reached 95%,
oxygen flow into the experimental cage was reduced to 5 l/min.
The exposure was terminated when the first convulsions
appeared. The inflowing gas was then changed back to air, and
pressure was reduced at a rate of 1 ATA/min. Latency to CNS-OT
was measured from the time O2 reached a level of 95% until
appearance of the first convulsions. The same setup was used
for exposure to pressure in each of groups 2–5.
4.4. Western blot analysis
The brain of each animal was thawed and homogenized with
SDS buffer (20% glycerol and 6% SDS in 0.12 M Tris buffer with
a pH of 6.8), centrifuged at 13,000g for 20 min at a tempera-
ture of 4 1C, and boiled for 10 min. The protein concentration
of the brain specimens was quantified by the Bradford
method (Bio-Rad Laboratories, Richmond, CA). Fifty micro-
gram total protein was loaded in each gel well. After blotting,
the membranes were incubated for 1 h in blocking solution
containing 5% skimmed milk in tris-buffered saline Tween-20
(TBST). The membranes were then washed briefly in TBST
and incubated overnight at 4 1C with the polyclonal IgG cross-
reactive to the antibodies of nitrotyrosine (NT) (Santa Cruz
Biotechnology, Santa Cruz, CA), nNOS (Cell Signaling Tech-
nology, Beverly, MA) and actin (Santa Cruz Biotechnology,
Santa Cruz, CA) diluted 1:1000. After three repeated washings
in TBST, the membranes were incubated at room tempera-
ture for 1 h with horseradish peroxidase-conjugated goat
anti-rabbit IgG in a 1:2000 dilution (Cell Signaling Technology,
Beverly, MA). The membranes were then developed to enhance
detection by chemiluminescence (Pierce Biotechnology, Inc.,
Rockford, IL) and exposed to X-ray film. Band densities were
quantified by scanning the films with a laser densitometer
(Image Master VDS-CL, Amersham Pharmacia Biotech, Uppsala,
Sweden). Levels of NT were expressed relative to actin.
4.5. Enzyme activity of SOD, CAT and GPX
The activity of SOD, catalase and GPX was measured using
commercially available assay kits (Cayman Chemical Com-
pany, Ann Arbor, MI).
4.6. Statistical analysis
Data are expressed as mean7standard deviation. Statistical
analysis was performed using one-way ANOVA. For a poster-
iori analysis we used Dunnett's test. Statistical significance
was defined as po0.05.
Disclosure statements
No conflicts of interest, financial or otherwise, are declared by
the authors.
Disclosure
The opinions and assertions contained herein are the private
ones of the authors, and are not to be construed as official or
as reflecting the views of the Israel Naval Medical Institute.
Grants
This study was supported by a research grant from the Israel
Defense Forces Medical Corps and the Israel MOD.
Acknowledgments
The authors thank Mr. R. Lincoln for skillful editing.
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