Vol. 91, Issue 3, 1193-1198, September 2001
Downregulation of nitric oxide in the brain of mice during
their hypoxic preconditioning
Guo-Wei
Lu and
Hong-Yan
Liu
Department of Neurobiology, Capital University of Medical Sciences,
Beijing 100054, China
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ABSTRACT |
An animal model of hypoxic
preconditioning was produced in mice by repeated exposure to
autohypoxic condition. The animals' tolerance times to hypoxia
were 1.7, 1.8, 2.1, and 2.3 times longer in runs 2,
3, 4, and 5, respectively, than that
in run 1, and their oxygen consumption and heart and
respiration rates were progressively and significantly slowed down
during the repetitive exposure to hypoxia. L-Arginine
concentration, nitric oxide (NO) synthase-positive cells, NO synthase
activity, and NO content in the whole brain and the subregions
telencephalon, diencephalon, and brain stem were significantly
increased during the first exposure and were, instead of continuing to
increase, significantly decreased in run 4 after the second
and third exposure. Tolerance times under the hypoxic condition were
significantly shortened and prolonged when preadministration of
L-arginine and its analog, respectively, was made. These
results indicate that NO in the brain is downregulated under condition
of hypoxic preconditioning and negatively involved in increased
tolerance to hypoxia.
L-arginine; nitric oxide synthase; N-nitro-L-arginine
| |
INTRODUCTION |
ISCHEMIC
PRECONDITIONING WAS first reported in 1986 by Murry et al.
(33) in terms of protection of the heart from subsequent lethal insult by previous exposure to a brief period of sublethal ischemia. A phenomenon called ischemic tolerance has
been demonstrated in the brain in vitro and in vivo since the 1990s
(3, 5, 12, 15-17, 24, 27, 35).
A unique model of a nonischemic but hypoxic method of
"preconditioning" was introduced in mice by Lu et al.
(28-30). Brief hypoxia was shown to increase
superoxide dismutase and adenosine in the brain during repetitive
exposure to air rebreathing from a small (125 ml) bottle, which is
called "hypoxic preconditioning" (11, 40, 41).
In contrast to these findings, the content or activity of some other
chemicals, namely lipid peroxides and glutamate, was decreased or
downregulated during the preconditioning (11, 37, 42).
Excessive release of excitatory amino acids is reported to be toxic to
neuronal cells, and nitric oxide (NO) is thought to be involved in
mediating the N-methyl-D-aspartate (NMDA)
neurotoxicity (6, 7, 17, 21, 22, 31). The present study
was designed to characterize whether the NO is downregulated or not
under the condition of hypoxic preconditioning.
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MATERIALS AND METHODS |
Experimental subject and hypoxic exposure.
Experiments were conducted at room temperature (18 ± 1°C) on
adult BALB/C mice of both sexes, weighing 16.0-22.0 g. The animals were anesthetized with a 1% solution of sodium pentobarbital (5.5 ml/kg ip) and randomly divided into three basic groups:
1) blank control group with no exposure to hypoxia
(H0), 2) hypoxia control group exposed to
hypoxia once (H1), and 3) hypoxic
preconditioning group exposed to hypoxia four or five times
(H4 or H5). For dynamic observation, groups
exposed to hypoxia two or three times (H2 or
H3) were also added.
The animal was placed into a 125-ml jar with fresh air, and the jar was
sealed with a rubber plug. The animal was removed from the jar as soon
as the first gasping breath appeared and was switched to another fresh
air-containing jar of similar volume. The duration of time between
subsequent exposures was variable within 30 s. The jar was
immediately hermetically sealed again. This procedure was performed
once (H1) and repeated two or three and four or five times
(H2 or H3 and H4 or H5,
respectively) (28-30).
At least three factors, lowering oxygen, increasing carbon dioxide, and
lowering atmospheric pressure, were thought to be involved in the
airless condition in the present procedure. It is generally recognized
that the main consequence is hypoxia, and the procedure is simply
described as "autohypoxia" (32). To make sure, the
carbon dioxide was absorbed by calcium hydroxide inside the jar, and
the atmospheric pressure was kept constant with a capsule, leaving only
hypoxia in the present study (Fig. 1A).
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Fig. 1.
Diagram showing apparatus for recording electrocardiogram
(ECG) and respiration movement (A) and examples of these
recordings (B). A: a, animal; b, balloon; e, ECG;
h, ECG recording; p, piezoelectrical crystal microphone; r, respiration
movement recording; t, tube for taking air from the jar. Dashed line,
metal net. B: left traces are ECG, and
right traces are respiration movement in runs 1 (I), 3 (III), and 5 (V). Nos. are time points (in
min) at which the records were taken in runs 1,
3, and 5.
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Tolerance time and O2 concentration determination.
The appearance of the first gasping was regarded as the tolerance limit
in each trial. The time period between the beginning of airtightness
and the appearance of the first gasping was termed "original duration
of tolerance" for each run. The standard tolerance duration in a
standard jar with an effective fresh air volume of 100 ml was
calculated as follows:
where T is the standard tolerance time (min),
T0 is the original tolerance time (min),
t0 is the starting time of sealing, t1 is the ending time of sealing, Ve
is the effective jar volume (ml), V0 is the original jar
volume (ml), Va is the animal's volume (ml),
Wa is the animal's body weight (g), and Da is
the animal's density determined from displacement Va and
measured body weight. The average Da was 0.94, ranging from
0.92 to 1.00 (28-30).
Microvolume (0.2 ml) of air in the tight jar was drawn via a tube
connected to the jar (Fig. 1A) at the end of each run when the first gasping occurred in the animal. A modified
Scholander-Roughton technique was used to determine the concentration
of oxygen in the sample (28).
Heart and respiration monitoring.
In addition to the observation of the gasping breath and general
behavioral responses, the animal's heart and respiration activity were
monitored during experimentation. An electrode was placed in the area
of the xiphoid process, and the other was in the corresponding level in
the back, and the signals were led to a modified electrocardiograph
(ECG). To record the animal's respiration movement, a piezoelectric
crystal microphone was connected to the capsule and connected to the
other lead of the ECG (Fig. 1A).
Cerebral arginine concentration measurement.
Animals were decapitated, and their heads were kept in 
10°C liquid
nitrogen right after hypoxic exposure of each group. The next day, the
whole brain was isolated and weighed. Pure water (1.5 ml) was added to
400 mg of whole brain. Samples were homogenized at <0°C and
centrifuged at 13,500 rpm for 45 min under 4°C temperature. After
the addition of 100 µl of 10% trichloroacetic acid to the supernatants, they were centrifuged at 13,500 rpm for 25 min under 4°C temperature. The supernatants were adjusted to pH 7-8 with 1 mol/l NaOH and were filtered with a 40-µm filter.
Twenty-two kinds of standard amino acids, including arginine, were
measured exactly and dissolved in 0.1 mol/l acetate buffer to receive 5 mmol/l of standard solution, which was respectively diluted to 1.0 mmol/l and 500, 100, 50, and 10 µmol/l of standard solution.
The standard supernatant samples were analyzed by HPLC with the
o-phthaldialdehyde precolumn derivatization technique. A
reverse-phase C18 column (150 × 3.9 nm, 4 µm, Nova
Pak, Waters) was used in the HPLC system (HP 1050 liquid chromatograph,
Hewlett-Packard). The mobile phase, which was composed of 0.1 mol/l
acetate buffer (pH 6.95) and methanol, was delivered at a flow rate of
0.8 ml/min by a high-pressure pump (HP 1050Q, Hewlett-Packard). After
derivative reaction with o-phthaldialdehyde, the samples (20 µl) were injected into the HPLC system by an autosampler and eluted
gradiently by HPLC. The contents of arginine in the sample were
detected by a fluorescence detector with excitation = 250 nm and
emission = 395 nm.
NADPH-diaphorase and NO synthase histochemistry
(43).
At the completion of experiments, the mice were anesthetized with a
lethal dose of 0.4% solution of sodium pentobarbital and were perfused
intracardially with phosphate-buffered saline. The animals were then
perfused with 200-300 ml of fixative containing 4%
paraformaldehyde. The animal's whole brain was removed and immersion
fixed in 30% solution of sucrose overnight.
On the next day, horizontal vibrotome sections (20 µm) of the brain
were cut; immersed and incubated in PBS solution containing 1 mmol/l
-NADPH, 1.2 mmol/l nitro blue tetrazolium, and 0.3% Triton X-100
(pH 8.0) at 37°C for 1 h; then transferred into PBS solution (pH
7.4) to end the reaction; and then counterstained with neutral red.
Photometric determination of NO synthase activity
(25).
Animals were decapitated, and their brains were removed and placed
quickly into liquid nitrogen. The animal's brain was taken from liquid
nitrogen and homogenized. Ten percent solution of trichloroacetic acid
in the supernatant was added and centrifuged to make it protein free.
The sample was then processed according to the description in the NO
synthase (NOS) activity kit and added, in the volume ratio of 20 to
100, to reaction solution containing 1.6 µmol/l Hb-O2,
200 µmol/l CaCl2, 1 mmol/l MgCl2, 100 µmol/l L-arginine, 11 µmol/l NADPH, 50 µmol/l
L-valine, and 40 mmol/l KPO3 (pH 7.2).
Absorption value was recorded for 3 min at 401 and 421 nm for every
30 s. NOS activity was expressed in moles per minute of NO and
calculated as follows
where a and b are absorption values at
30 s at 401 and 421 nm, respectively, and a' and
b' are values at 90 s at 401 and 421 nm, respectively.
Fluorometric determination of NO (34,
36).
The animal's whole brain and subregions of telencephalon,
diencephalon, brain stem, and cerebellum were taken out, and 2 ml of
PBS solution were added and homogenized under ice bath. One-half milliliter of the sample was kept for measurement of protein. Trichloroacetic acid (10%) was added to the remaining sample and centrifuged to make it protein free. One-half milliliter of 0.04% 4-hycoumarin reagent was added to 1 ml of supernatant in a 10-ml stoppered test tube. The tube was left in an ice bath for 5 min. To the
reaction mixture was added 0.1 ml of 8% sodium sulfate, which was left
for 10 min at room temperature, and reduced mixture was made alkaline
with 1.0 ml of 1.5 mol/l NaOH and left for 10 min at room temperature.
The method is based on the nitrosation of 4-hydroxycoumarin in acidic
medium and subsequent reduction to 3- amino-4-hydroxycoumarin, which
is fluorescent in alkaline medium. The fluorescent intensity is
proportional to the nitrite concentration in the range of 3 ng/ml to 1 µg/ml in the sample solution, with a relative standard deviation of
0.5% (50 ng/ml) (34). The fluorescence intensity was
measured with excitation at 347 nm and emission at 453 nm. A standard
curve was made with NaNO2. NO values were expressed in
picomoles per milligram of protein for subregions of the brain.
Administration of L-arginine and its analog.
Mice were divided randomly into groups of L-arginine,
L-arginine analog, and normal saline.
L-Arginine (50 mg/kg),
N-nitro-L-arginine (L-NNA; 50 mg/kg), and normal saline (90 mg/kg) were administrated intraperitoneally to the three respective groups. Thirty minutes later,
animals were exposed to hypoxia repeatedly as shown in the model. The
standard tolerance times of runs 1-4 were compared correspondingly among the three groups.
Statistical analysis.
ANOVA and Duncan's test from the SYSTAT program were used for analysis
of experimental data. A level of 0.05 or less was accepted as an
indicator of significance.
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RESULTS |
During the preconditioning, the animals' respiration gradually
quickened, cyanosis gradually increased, and finally spasmlike activity
and gasping breath appeared in the first run of exposure to hypoxia.
Similar manifestations were evident during the second run. Starting
with the third run, the animal remained quiet most of the time, and its
respiration become slow, deep, but regular in pattern (Fig.
1B). Cyanosis become more apparent in the fourth run, and
the eyeballs showed a black-violet color.
Changes in tolerance time and chamber oxygen level.
The increment of each run in standard tolerance time was progressive
when the animals were under anesthesia, and the preconditioning procedure was taken at room temperature of 18 ± 1°C. The
tolerance time was 1.7, 1.8, 2.1, and 2.3 times longer in runs
2, 3, 4, and 5, respectively,
than in run 1 (Fig.
2A). The oxygen concentration inside the jar at the moment of appearance of the first gasping was
6.6% in run 1 and progressively decreased to 3.2% in
run 5, which was 49% of that in run 1 (Fig.
2B).
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Fig. 2.
Tolerance time (A), ending oxygen
concentration in the jar (B), and heart (HR) and respiration
rate (RR; C) in different runs of exposure. Values are
means ± SD; n = 15 (A, B)
and 20 animals (C). c/min, Cycles/min (beats/min for heart
rate and breaths/min for respiration rate); HR-i, initial HR; HR-e,
ending HR; RR-i, initial RR; RR-e, ending RR. Significant difference
compared with run 2 (A) and with corresponding
value in run 1 (C), * P < 0.05;
significant difference compared with preceding run (A and
B) and corresponding value in run 1 (C), ** P < 0.01.
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The rhythm of ECG was kept regular all the way from run 1 through 5 (Fig. 1B). The average initial heart
rate at the beginning of each run progressively decreased from 744 beats/min in run 1 to 180 beats/min in run 5, and
the ending one at the first grasping was reduced from 469 beats/min in
run 1 to 157 beats/min in run 5, which was
one-fifth of initial rate in run 1 (Fig. 2C).
The same pattern was shown in the rhythm of respiration (Fig.
1B). The average initial (ending) respiration rate also
decreased progressively from 315 breaths/min (300 breaths/min) in
run 1 to 96 breaths/min (78 breaths/min), which was also
one-fifth of the initial rate in run 1 (Fig. 2C).
Changes in cerebral L-arginine content.
As shown in Fig. 3, the content of
L-arginine in whole brain was 347.182 ± 49.354, 430.864 ± 41.770, 307.303 ± 55.690, 296.033 ± 41.211,
and 293.080 ± 51.830 nmol/g in groups H0,
H1, H2,
H3, and H4, respectively.
The content significantly increased in group H1,
progressively decreased in groups
H2-H4, and even was
significantly less than that in group H0.
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Fig. 3.
L-Arginine (L-Arg) contents of
whole brain in different runs. H0-H4,
hypoxia exposure for 0-4 times, respectively. Values are
means ± SD; n = 10 animals for each group.
Significant difference compared with H0:
* P < 0.05 and ** P < 0.01;
significant difference compared with H1,
# P < 0.05.
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Changes in NOS-positive neurons and NOS activity in the brain.
No apparent difference in number and shape of NOS-positive neurons in
the cerebral cortex was seen among groups H0,
H1, and H4. However, the
diameter of processes of positive neurons was clearly more enlarged in
group H1 compared with both groups
H0 and H4, the intensity
of cell stain in group H1 was more positive than
that in both groups H0 and
H4, and no apparent difference was shown between
the latter two groups (Fig.
4A). The number of
NOS-positive neurons in the hippocampus was 5, 42, and 39 on average in
groups H0, H1, and
H4, respectively. Instead of continuing to
increase, the number tended to decrease in group
H4 (Fig. 4B).
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Fig. 4.
Nitric oxide synthase (NOS)-positive neurons on hemispheres
(A) and hippocampus (B) in groups
H0 (1), H1
(2), and H4 (3).
Magnification ×400 (A) and ×100 (B).
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NOS activity in the whole brain dramatically increased in group
H1 and, instead of continuing to increase, it
decreased significantly and tended toward the control level in
group H4 (Fig.
5A).
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Fig. 5.
NOS activity (A), nitric oxide content in
whole brain (B), and different brain subregions
(C) in groups H0,
H1, and H4. Values are
means ± SD; n = 16 (A), 15 (B), and 10 animals (C). Significant difference
compared with H0: * P < 0.05 and
** P < 0.01; significant difference compared with
H1: # P < 0.05 and
## P < 0.01.
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Changes in cerebral NO content.
Similar to the changes in NOS activity, the content of NO in whole
brain also dramatically increased in group
H1 and significantly decreased to control level
in group H4 (Fig. 5B). The value of NO content was 6.294 ± 2.337, 18.879 ± 10.491, and
8.280 ± 2.130 pmol/mg protein in groups
H0, H1, and
H4, respectively.
Similar changes were shown in the subregion of the telencephalon,
diencephalons, and brain stem (Fig. 5C). The NO content in
these subregions markedly increased in group H1
and tended to reduce to the group H0 level in
group H4 (Fig. 5C).
Effects of extraneous arginine and its analog.
Behaviorally, more severe responses and significant shortening in
tolerance time were shown during the second, third, and fourth run of
exposure in the arginine group (Fig. 6).
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Fig. 6.
Standard tolerance time of different runs in group
control, L-Arg, and L-Arg analog. Values are
means ± SD; n = 15 animals for each group.
Significant difference compared with corresponding value in preceding
run, * P < 0.05; significant difference compared
with control value within run, # P < 0.05.
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However, less and less behavioral manifestation and a significant
prolongation in tolerance time were shown during runs
1-4 when the analog of arginine, L-NNA, was given
to the animals before the exposure to hypoxia (Fig. 6).
| |
DISCUSSION |
Similar to our laboratory's previous findings
(28-30), the present study shows that the animal is
able to tolerate progressively lowered levels of oxygen and to keep
alive in a lowered state of life activity for longer and longer time
periods during repeated exposure to hypoxia. The progressive lowering
of life activity, as shown by slowed oxygen consumption and heart and
respiration rate, seems to be beneficial to the progressive increase in tolerance.
NO is a putative neurotransmitter in the neuronal systems and is
synthesized from L-arginine by the enzyme NOS (2,
19). Based on existing knowledge, it is most likely that
neuronal NOS (nNOS) is responsible for the observed phenomenon
(12). However, it is not possible to rule out the
contribution of the endothelial isoform.
NO may act as both a neuroprotective and a neurodestructive agent in
the hypoxic and ischemic injuries (1, 18, 38). NO
donors or NO produced by constitutive nNOS limit apoptosis induced by trophic factor deprivation in PC12 cells and primary neurons
(13, 14, 23, 26). NO from constitutive hippocampal NOS may
be involved in the neuroprotection afforded by preconditioning (4), and NO production and activity are critical to the
induction of ischemic tolerance in vivo (15).
In contrast, NO is able to lead to neuronal cell death when it is
produced in excess (8-10, 18, 39). Several reports of increased NO production after ischemia have been published
(22, 31). Stroke damage is diminished in nNOS/ mice
(20, 21), the damage is also reduced by nNOS inhibitors
(22), and nNOS activation is linked to neural damage
(12).
What we have seen in the present study is consistent with these
reports. The content of the NO donor L-arginine,
NOS-positive cells, NOS activity, and NO content in the brain increased
initially in run 1 and then decreased in run 4 after repeated hypoxic exposure, whereas tolerance increased steadily
with each trial. The decreased NOS activity and NO content seem to be
related to the increase in tolerance to hypoxia.
The downregulation of these indexes may thus be beneficial to the
increase in the animals' tolerance to hypoxia. This seems to be
confirmed by the effect of preadministration of extraneous L-arginine and its analog. The animals' tolerance time
under the condition of hypoxia is significantly shortened and prolonged by administration of the NO donor L-arginine and the
L-arginine analog L-NNA, respectively.
NMDA neurotoxicity is markedly decreased after treatment with NOS
inhibitors and in cultures from nNOS-deficient mice (8, 10). The increase in tolerance to hypoxia by arginine analog administration and the negative correlation between the increment in
tolerance and decrease in NOS-positive neurons and activity, as well as
in NO production, might be related to the decrease in NMDA neurotoxicity.
NO itself may not be toxic and becomes toxic only when it reacts with
superoxide and is converted to peroxynitrite (1). Instead
of a continuing increase in lipid peroxides during hypoxic preconditioning, its content became less and tended to reach control level (11). In addition, the increased activity of
superoxide dismutase during repetitive exposure to hypoxia
(11) might also be beneficial to the reduction of
peroxides. The decrease in NO toxicity may also be involved in the
development of hypoxic preconditioning and tolerance to hypoxia.
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ACKNOWLEDGEMENTS |
This work was supported by the National Natural Science Foundation
of China (Grant 39670871) and Beijing Natural Science Foundation (Grant 7962009).
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FOOTNOTES |
Address for reprint requests and other correspondence: G.-W.
Lu, Dept. of Neurobiology, Capital Univ. of Medical Sciences, You An
Men Wai St., Beijing 100054, China (E-mail:
gwlu{at}cpums.edu.cn).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 15 December 2000; accepted in final form 30 April 2001.
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J APPL PHYSIOL 91(3):1193-1198
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Copyright © 2001 the American Physiological Society
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ABSTRACT
INTRODUCTION
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