(Stroke. 1999;30:669-677.)
© 1999 American Heart Association, Inc.
Original Contributions |
Nitric Oxide Production in the CA1 Field of the Gerbil Hippocampus After Transient Forebrain Ischemia
Effects of 7-Nitroindazole and NG-Nitro-L-Arginine Methyl Ester
From the Department of Anesthesiology and Resuscitology, Ehime University School of Medicine, Ehime, Japan.
Correspondence to Naoto Adachi, MD, PhD, Department of Anesthesiology and Resuscitology, Ehime University School of Medicine, Shitsukawa, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan.
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Abstract |
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Background and Purpose—The present study was designed to examine the time course of nitric oxide (NO) production and the source of NO in the CA1 field of the gerbil hippocampus after transient forebrain ischemia.
Methods—The production of NO in the CA1 field of the hippocampus after transient ischemia was monitored consecutively by measuring total NO metabolites (NOx-, NO2- plus NO3-) with the use of brain microdialysis. 7-Nitroindazole (7-NI) and NG-nitro-L-arginine methyl ester were used to dissect the relative contributions of neuronal NO synthase and endothelial NO synthase to the NO production. The histological outcomes of 7-NI in 5- and 10-minute global ischemia were also evaluated.
Results—The production of NO in the CA1 field of the hippocampus after ischemia was dependent on the severity of ischemia. Ischemia for 2 or 5 minutes did not induce a significant increase in NOx- levels in the CA1 field of the hippocampus after reperfusion, whereas the 10- and 15-minute ischemias produced significant and persistent increases in NOx- levels. 7-NI did not inhibit the basal NOx- levels and showed no effects on NOx- levels after 5 minutes of ischemia. However, it completely inhibited the increased NOx- levels after 10 or 15 minutes of ischemia. 7-NI provided minor neuroprotection in 5 minutes but not in 10 minutes of global ischemia.
Conclusions—The increased NO level in the CA1 field of the hippocampus after ischemia is produced mostly by neuronal NO synthase, whereas the basal NO level mainly originates from endothelial NO synthase. The observed neuroprotective effect of 7-NI in 5-minute global ischemia in gerbils may not be due to neuronal NO synthase inhibition by this drug.
Key Words: cerebral ischemia • microdialysis • nitric oxide • gerbils
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Introduction |
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The role of nitric oxide (NO) in the pathogenesis of ischemic brain damage has been controversial.1 2 3 4 Evidence has accumulated that NO may play either a neurotoxic or a neuroprotective role after cerebral ischemia. As a result of the multiple and contrasting actions of NO as well as the different sources of NO synthase (NOS), modulations of NO in ischemic brain have been shown to produce a variety of outcomes.5 6 Both neuronal NOS (type I; nNOS) and inducible NOS (type II; iNOS) activity have been proposed to be detrimental to the ischemic brain, whereas endothelial NOS (type III; eNOS) activity might be protective.3 4 However, little information is available on the contributions of 3 isoforms of NOS to NO production in the brain after ischemia. Since the effects of NO on the ischemic brain are thought to be dependent on the stage of the ischemic process and the sources of its production,3 it is necessary to elucidate the time course of NO production associated with transient cerebral ischemia and the source of NO in the postischemic brain.
Certain brain regions, especially the CA1 region of the hippocampus, are selectively vulnerable to brief periods of global ischemia.7 8 9 The mechanism underlying this vulnerability is unknown, and the involvement of NO in the process of delayed neuronal death observed in this region has not been clarified. Although several studies showed that 7-nitroindazole (7-NI), a selective nNOS inhibitor, protected against ischemia-induced hippocampal damage in the gerbil and rat,10 11 12 NO production was not measured in these studies. In the present study we measured the amount of total NO metabolites (NOx-, NO2- plus NO3-) by brain microdialysis to monitor the production of NO in the CA1 field of the hippocampus in a gerbil model of cerebral ischemia, and we examined the relationship between NO production and the severity of ischemia. We also evaluated the effects of 7-NI and NG-nitro-L-arginine methyl ester (L-NAME) on NO production to determine the relative contributions of nNOS and eNOS to NO production. Furthermore, the histological outcomes of 7-NI were examined in 5- and 10-minute global ischemia.
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Materials and Methods |
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Animals
Male Mongolian gerbils (weight, 60 to 80 g) (Seiwa Experimental Animals, Fukuoka, Japan) were housed in a room controlled at 23±2°C with controlled lighting conditions (12 hours light/12 hours dark). Food and water were provided ad libitum. All animal use procedures were approved by the Committee on Animal Experimentation at Ehime University School of Medicine, Ehime, Japan.
Experiment 1: Effects of Transient Forebrain Ischemia on
NO Production
The time courses of NO production in the CA1 field of
the hippocampus after various durations of ischemia were
examined. Thirty-five gerbils were divided into 6 groups. Seven animals
served as controls and underwent sham operations without carotid artery
occlusion. In the other groups, ischemia was induced for 2, 5,
10, 15, or 20 minutes (n=7, n=6, n=5, n=5, and n=5, respectively).
The gerbils were anesthetized and maintained with a mixture of
3% halothane and nitrous oxide/oxygen (1:1) with the use of a face
mask. A midline neck incision was made, and both common carotid
arteries were exposed and separated carefully from adjacent nerves and
tissues. Silk threads (4-0) were looped around these arteries. After
the animal was placed in a stereotaxic
apparatus (David Kopf Instruments), the skull was
exposed through a midline scalp incision. Two small burr holes were
drilled to insert probes for brain temperature measurement and for
microdialysis. The dura was carefully incised. A thermocouple needle
probe was inserted in the right brain through a burr hole. The tip of
the thermocouple probe was positioned 
3 mm anterior and 2
mm lateral to the bregma and 2 mm below the brain surface. A guide
cannula (A-I-8, Eicom) was perpendicularly implanted into the right
hippocampus (2 mm posterior and 2 mm lateral to the bregma
and 1.4 mm below the brain surface). The administration of nitrous
oxide was then discontinued, and the anesthesia was
maintained with 2% halothane delivered with oxygen at 2 L/min
throughout the experiment. The microdialysis probe (A-I-8-01, 1-mm-long
dialysis membrane, OD 0.22 mm; molecular weight cutoff at 50 000;
Eicom) was inserted through the guide cannula into the CA1 field of the
right hippocampus and perfused with Ringer's solution at a constant
rate of 2 µL/min with a microinjection pump. After a stabilization
period of 90 minutes, dialysates were collected every 15 minutes into
microtubes on ice, then stored at -80°C until analysis. The
threads around both common carotid arteries were pulled by 8-g weights
to occlude the circulation. After various durations of
ischemia, the threads were cut and removed to restore the blood
flow.13 Rectal and brain temperatures were maintained at
37.0°C to 37.5°C with heating lamps during the experimental
period.
In a separate group of animals, mean arterial blood pressure (MABP) and arterial blood gases were checked during ischemia and reperfusion. The animals were anesthetized and maintained as described above. A polyethylene catheter (PE-10; Becton Dickinson) was inserted into the femoral artery for monitoring MABP and collecting blood samples. The bilateral common carotid arteries were dissected free by a ventral neck incision and occluded with aneurysm clips. After various durations of ischemia (2, 5, 10, 15, and 20 minutes), the clips were removed. The rectal temperature was monitored and kept at 37.0°C to 37.5°C during the experimental period.
Experiment 2: Effects of 7-NI and L-NAME on NO Production
The effects of 7-NI and L-NAME on NO production in the
CA1 field of the hippocampus after ischemia were evaluated.
Eighty-three gerbils were divided into 16 groups: nonischemic
peanut oil or 7-NI group; nonischemic saline or L-NAME group;
ischemic (5, 10, and 15 minutes) peanut oil or 7-NI groups; and
ischemic (5, 10, and 15 minutes) saline or L-NAME groups. Each
group contained 5 to 7 gerbils. The animals were prepared as described
above, and an intraperitoneal catheter was inserted
into the abdominal space for drug administration. 7-NI (80 mg/kg) or
L-NAME (60 mg/kg) was injected by means of the catheter. In the
5-minute ischemic groups, ischemia was induced 30
minutes after the injection of 7-NI or L-NAME. In the 10- and 15-minute
ischemic groups, 7-NI or L-NAME was injected
intraperitoneally immediately after the onset of
ischemia. In the ischemic control groups, the animal
was given peanut oil or saline. 7-NI was suspended in peanut oil (40
mg/mL) with sonication. L-NAME was dissolved in saline (10 mg/mL).
Experiment 3: Histology
The histological outcome of the 7-NI
administration was examined. After anesthesia, a catheter
was inserted into the abdominal space for drug administration.
Twenty-seven gerbils were divided into 5 groups: sham-operated group
(n=5); 5-minute ischemic oil or 7-NI group (n=6 for each); and
10-minute ischemic oil or 7-NI group (n=5 for each). In the
5-minute ischemic groups, ischemia was induced 30
minutes after the injection of oil or 7-NI (80 mg/kg). In the 10-minute
ischemic groups, oil or 7-NI was injected immediately after the
onset of ischemia. The rectal temperature was monitored and
maintained at 37.0°C to 37.5°C throughout the experiment.
Determination of NOx- in
Dialysate
The amount of NOx-
(NO2- plus
NO3-) in the dialysates was
determined by the 2,3-diaminoaphthalene (DAN) method with slight
modification.14 15
NO3- in the dialysate was first
reduced to NO2- by nitrate
reductase, and then the amount of
NO2- was determined. Briefly,
an aliquot of the dialysate was incubated with 25 µmol/L
ß-nicotinamide adenine dinucleotide phosphate (reduced
form [NADPH]), 25 µmol/L flavin adenine
dinucleotide, and 10 mU of nitrate reductase in a final
volume of 50 µL of 20 mmol/L Tris-HCl buffer (pH 7.6) for 60
minutes at room temperature while protected from light. To terminate
the reaction, 150 µL of distilled deionized water was added. Then 20
µL of DAN solution (31.6 µmol/L in 0.62 mol/L HCl, prepared
from 31.6 mmol/L stock solution in
N,N-dimethylformamide [DMF]) was added and
mixed immediately. After a 10-minute incubation at room temperature in
the dark, the reaction was terminated with 300 µL of 93.32
mmol/L NaOH. The fluorescence was measured at an excitation
wavelength of 365 nm and an emission wavelength of 405 nm with a
spectrofluorometer (FP-777, JASCO). A standard curve prepared with
solutions of known NaNO2 concentrations (from
0.25 to 2 µmol/L) was run in each assay. To determine the rate
of NO3- to
NO2- conversion by nitrate
reductase, a series of solutions of known NaNO3
concentrations (from 0.25 to 2 µmol/L) was also assayed. The
total NO2- concentration
(representing NO2-
plus NO3-) in the dialysate was
calculated from the standard curve. The level of
NO2- alone in the dialysate was
undetectable, and the NO3- to
NO2- conversion was >90%. The
detection limit of this method was 0.1 µmol/L, equivalent to
1 pmol of NO3- in 10 µL of
dialysate.
The in vitro recovery of NO2- and NO3- across the 1-mm dialysis probe was determined by immersing the microdialysis probes in a Ringer's solution containing 10 µmol/L NaNO2 or 10 µmol/L NaNO3 at 37°C. The probe was perfused with Ringer's solution at a rate of 2 µL/min, and fractions were collected every 15 minutes for the analysis of NO2- and NO3- levels.
Histological Analysis
All animals in the second experiment and those subjected to 15-
and 20-minute ischemia in the first experiment were not allowed
to recover from anesthesia at the end of microdialysis
experiment. The animals were perfused transcardially with 0.9% saline
and then with 10% buffered formalin solution. The brains were removed,
and the location of the microdialysis probe was microscopically
determined.13 The other animals in the first experiment
and those in the third experiment were allowed to recover from
anesthesia. Seven days later, the animals were
anesthetized with pentobarbital again and transcardially
perfusion-fixed with the same procedure as described above. Paraffin
sections, 5 µm thick, were stained with hematoxylin and eosin.
The number of intact CA1 neurons per 1-mm length of stratum
pyramidale was counted in the same level of coronal
sections (2.0 mm posterior to bregma).
Chemicals and Reagents
DAN, DMF, NaOH, HCl, NaNO2, and
NaNO3 were obtained from Wako Pure Chemical
Industries. 7-NI, L-NAME, peanut oil, nitrate reductase (EC 1.6.6.2,
from Aspergillus species), NADPH, and flavin adenine
dinucleotide were purchased from Sigma.
Statistical Analysis
The data from microdialysis are expressed as mean±SD of
percentage of basal levels (the mean of 2 values before
ischemia or drug administration). Statistical significance was
determined by ANOVA, followed by Fisher's protected least significant
difference test or by Student's t test. The statistical
analysis of histological data was performed
with ANOVA followed by Scheffé's test. Comparisons among the 6
groups of MABP and blood gases were analyzed by ANOVA followed
by Scheffé's test. A probability value of <0.05 was regarded as
significant.
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The Table
shows MABP and blood gas
parameters during ischemia and reperfusion. There
were no significant differences in MABP, pH,
PaCO2, or
PaO2 among any of the groups before
ischemia and 30 minutes after reperfusion. MABP was markedly
increased during ischemia in all ischemic groups, but
there were no significant differences among the ischemic
groups.
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The basal level of NOx- (NO2- plus NO3-) in the dialysate in the hippocampus was 0.26±0.10 µmol/L (n=35). The in vitro recoveries of NO2- and NO3- across the 1-mm dialysis membrane were estimated to be 14.6±2.0% and 13.8±2.5%, respectively (n=3).
Figures 1 and 2 show the changes in
NOx- levels in the dialysate in
the hippocampus after various durations of ischemia. In the
control group, the levels of
NOx- in dialysates in the
hippocampus were stable throughout the experimental period. During
ischemia, the NOx-
levels were decreased in all ischemic groups. In the 2- and
5-minute ischemic groups, the levels of
NOx- returned to the basal
level 15 minutes after reperfusion and were not significantly different
from those in the control group throughout the reperfusion period
(Figure 1). In the 10- and 15-minute ischemic groups,
the levels of NOx- were
significantly increased 30 minutes after reperfusion, and the elevation
continued for 180 minutes after reperfusion. The levels at 45 and 60
minutes (233% and 236% of the basal level, respectively) and at 150
minutes (261% of the basal level) of reperfusion in the 15-minute
ischemic group were markedly greater than those of the other
ischemic groups at the corresponding time points (Figure 2). In the 20-minute ischemic group, the increases in
NOx- levels were observed only
at 45 and 60 minutes of reperfusion.
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As shown in Figure 3, in
nonischemic gerbils, 7-NI (80 mg/kg) had no effect on the
levels of NOx- in the
hippocampus. L-NAME (60 mg/kg) decreased the levels of
NOx- to 50% of the control
level.
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Figure 4 shows the effects of 7-NI (A)
and L-NAME (B) on NOx- levels
after 5-minute ischemia. 7-NI had no effect on the
NOx- levels before
ischemia. L-NAME decreased the levels of
NOx- 30 minutes after the
intraperitoneal injection. During the
ischemic period for 5 minutes, the levels of
NOx- were decreased in all
ischemic groups. In the ischemic oil and saline groups,
the levels of NOx- were not
significantly different from those in the corresponding sham control
groups throughout the reperfusion period. 7-NI had no effect on the
levels of NOx- during
reperfusion, whereas L-NAME decreased the
NOx- levels to 50% of the
sham control level.
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As shown in Figures 5A and 6A, the increased levels of
NOx- after 10- or 15-minute
ischemia were completely inhibited by 7-NI. L-NAME abolished
the increased levels of NOx-
after 10-minute ischemia and decreased the
NOx- levels after 15-minute
ischemia (Figures 5B and 6B). The levels from 30
to 60 minutes after reperfusion in the 15-minute ischemic
L-NAME group were higher than those in the saline-injected sham group
but significantly lower than those in the ischemic saline group
(Figure 6B).
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Figure 7 shows the effects of 7-NI on the
neuronal density in the CA1 region of the hippocampus 7 days after 5
and 10 minutes of ischemia. The number of intact CA1 neurons
was significantly lower in the ischemic oil and 7-NI groups
than in the sham-operated group. In the 5-minute ischemic 7-NI
group, the number of intact CA1 neurons was significantly greater than
that in the 5-minute ischemic oil group. There were no
significant differences in the number of intact CA1 neurons between the
10-minute ischemic oil group and the 10-minute ischemic
7-NI group.
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Discussion |
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NO is an unstable molecule that is easily degraded into NO2- and NO3-.16 The dynamic evaluation of NO production is thus thought to be difficult. Several studies used microdialysis to detect NO2- and NO3- as a measure of NO production in the brain.17 18 19 20 21 It has been shown that the levels of NOx- in the dialysate can serve as a reliable indicator of NO production in the in vivo brain. In the present study we detected NOx- levels in dialysate by using a modified DAN method, which is simple, rapid, and sensitive and does not require any special equipment.
It is clear that cerebral ischemia/reperfusion results in an increased production of NO.20 21 22 23 NO production in the brain depends on substrate availability, NOS levels, and NOS activity. In the present study we found that there was a significant decrease in NO production in the CA1 field of the hippocampus during ischemia. This is consistent with the requirement of oxygen for the formation of NO and L-citrulline from L-arginine. Furthermore, a decrease in shear stress on the endothelial wall could mediate the decrease in NO levels during ischemia.24
After reperfusion, the increase in NO production may be attributable to the resupply of oxygen and the activation of constitutive NOS by an intracellular calcium elevation during reperfusion.25 Reperfusion may also lead to an increase in shear stress and the restoration of eNOS activity.24 In addition, eNOS and nNOS levels have been reported to increase rapidly in the rat brain after focal cerebral ischemia.26 27 In the present study NO production was not increased after 2- and 5-minute ischemia. Significant increases were observed after 10 and 15 minutes of ischemia. However, the extent of NO production after 20 minutes of ischemia was decreased. These findings suggest that the threshold for the increase in NO production in the CA1 field of the hippocampus may be higher than 5-minute ischemia and lower than 10-minute ischemia and that the capacity of the CA1 field to produce NO may be limited (a plateau effect).
In this study 7-NI did not reduce the basal NO production and exerted no effect on NO production after 5 minutes of ischemia. L-NAME, in contrast, decreased the basal NO production and NO production after 5 minutes of ischemia. L-NAME is a nonspecific inhibitor, acting on both nNOS and eNOS,28 whereas 7-NI has a high specificity for nNOS in vivo.29 30 31 32 We therefore believe that the basal NO and the NO after 5 minutes of ischemia are mainly produced by the action of eNOS. It was recently demonstrated that long-term potentiation in the CA1 region, which is mediated by NO, still exists in mice that lack nNOS33 and that eNOS immunoreactivity is selectively concentrated in the CA1 region of the hippocampus,34 suggesting that eNOS is responsible for most of the basal NO level in the hippocampus. Other studies, by using an ex vivo assay of NOS, indicated that 7-NI inhibited NOS in the hippocampus.35 36 The divergent results may be due to the fact that the ex vivo assay of NOS is not necessarily a reliable index of actual NO production in vivo and has less site specificity than microdialysis. However, since intraperitoneal administration of 7-NI even at high doses usually inhibits ex vivo total NOS activity in the supernatant fraction (where nNOS is thought to be more heavily concentrated than eNOS) by only 50% to 60%,35 36 it is possible that 7-NI does not produce complete inhibition of nNOS. Thus, 7-NI may not be capable of inhibiting nNOS sufficiently to inhibit basal activity but only evoked increases in activity.
Since the increased NO production after 10- or 15-minute ischemia was completely inhibited by 7-NI in the present study, NO production may be due to the action of nNOS. In the hippocampus, nNOS exists mainly in the interneurons and astrocytes,34 37 although it was also found in the CA1 pyramidal neurons.38 However, the interneurons and astrocytes are resistant to ischemia and presumably not affected by 5 minutes of ischemia.39 Therefore, the sustained increase in NO production after 10- or 15-minute ischemia may be caused by the activation of nNOS in interneurons and astrocytes. In the present study 7-NI abolished the increased NO level in these groups but did not decrease the NO amount to the level below the basal level, implying that eNOS still partly contributes to an increase in NO after 10- and 15-minute ischemia. However, L-NAME did not abolish the increased NO production after 15 minutes of ischemia. The lack of complete abolition of the increased NO production may be attributed to the limited inhibitory ability of L-NAME.
It is well known that 5-minute ischemia produces complete destruction of the CA1 pyramidal cells in the gerbil.8 Because we did not observe an increase in NO production in the present 5-minute ischemic group, it is unlikely that NO produced from nNOS is involved in the vulnerability of the CA1 cells of the hippocampus after transient ischemia. Several investigators reported that 7-NI protected against ischemia-induced delayed neuronal death of the CA1 pyramidal neurons.10 11 12 We also found in the present study that 7-NI was neuroprotective in 5-minute global ischemia. Because NO production was not increased after 5-minute ischemia and 7-NI did not exert an influence on NO production after 5 minutes of ischemia, the observed neuroprotective effect of 7-NI may be derived from other unknown mechanisms rather than nNOS inhibition. However, there is a possibility that a large but short burst of NO at the start of reperfusion may occur in the 5-minute ischemic gerbils, while we failed to detect an increase in NO because of the long sampling intervals of the microdialysis technique. In addition, in the present study NO production was measured only in the early stage of reperfusion. Since the hippocampal CA1 neurons after ischemia deteriorate very slowly and NOS catalytic activity increases 4 days after 5-minute ischemia in this model,40 we cannot exclude the possibility that NO produced from iNOS in the late stages of the damage is involved in the mechanism of brain damage after global ischemia.
In the present study 7-NI inhibited the increased NO
production after 10 minutes of ischemia, but it had no
neuroprotective effect. However, failure to demonstrate a
neuroprotective effect with 7-NI in 10-minute global ischemia
may be a result of inadequate absorption, because 7-NI was given after
the onset of ischemia in the 10-minute ischemic group.
Thus, toxic amounts of NO could be produced during ischemia or
early reperfusion and would not be inhibited in the 10-minute
ischemic protocol. Because 10-minute ischemia may be
too severe to evaluate its protection against ischemic injury
and the dosing regimens of our present study may be improper, the
question of whether the increased NO level after 10 and 15 minutes of
ischemia is beneficial or detrimental to the brain cannot be
answered by the present study. Although damage to the hippocampal
CA1 sector after 5 minutes of ischemia occurs extremely slowly,
the progression of damage becomes faster when the ischemic
period is prolonged.8 The increased NO production
after 10 and 15 minutes of ischemia may be involved in the
progression of damage, because high concentrations of NO are toxic and
interact with superoxide to produce the highly toxic peroxynitrite
anion (ONOO-).41 According to the
in vitro recovery of NO3-
observed in the present study, the extracellular concentrations of
NO in the hippocampus after 10 and 15 minutes of ischemia are
estimated to be 3.0 to 4.5 µmol/L. These concentrations are
believed to be sufficient to elicit peroxynitrite
formation.42 Therefore, an excessive production of
NO may exert acutely lethal influences on the CA1
neurons.43
In conclusion, the data in the present study indicate that NO production in the CA1 field of the hippocampus after ischemia depends on the severity of ischemia. The data also indicate that mainly eNOS contributes to the basal NO production, whereas nNOS is mostly responsible for the increased NO production after ischemia. The observed neuroprotective effect of 7-NI in 5-minute global ischemia in gerbils may be due to other unknown mechanisms rather than nNOS inhibition by this drug.
Received September 15, 1998; revision received December 3, 1998; accepted December 17, 1998.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
1. Choi DW. Nitric oxide: foe or friend to the injured brain? Proc Natl Acad Sci U S A. 1993;90:9741–9743.
2. Dawson TM, Snyder SH. Gases as biological messengers: nitric oxide and carbon monoxide in the brain. J Neurosci. 1994;14:5147–5159.[Abstract]
3. Iadecola C. Bright and dark sides of nitric oxide in ischemic brain injury. Trends Neurosci. 1997;20:132–139.[Medline] [Order article via Infotrieve]
4.
Samdani AF, Dawson TM, Dawson VL. Nitric oxide
synthase in models of focal ischemia. Stroke. 1997;28:1283–1288.
5. Panahian N, Yoshida T, Huang PL, Hedleywhyte ET, Dalkara T, Fishman MC, Moskowitz MA. Attenuated hippocampal damage after global cerebral ischemia in mice mutant in neuronal nitric oxide synthase. Neuroscience. 1996;72:343–354.[Medline] [Order article via Infotrieve]
6.
Kirsch JR, Bhardwaji A, Martin LJ, Hanley DF,
Traystman RJ. Neither L-arginine nor L-NAME affects neurological
outcome after global ischemia in cats. Stroke. 1997;28:2259–2265.
7. Smith ML, Auer RN, Siesjö BK. The density and distribution of ischemic brain injury in the rat following 2–10 min of forebrain ischemia. Acta Neuropathol (Berl). 1984;64:319–332.[Medline] [Order article via Infotrieve]
8. Araki T, Kato H, Kogure K. Selective neuronal vulnerability following transient cerebral ischemia in the gerbil: distribution and time course. Acta Neurol Scand. 1989;80:548–553.[Medline] [Order article via Infotrieve]
9. Kirino T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 1982;239:57–69.[Medline] [Order article via Infotrieve]
10. O'Neill MJ, Hicks C, Ward M. Neuroprotective effects of 7-nitroindazole in the gerbil model of global cerebral ischemia. Eur J Pharmacol. 1996;310:115–122.[Medline] [Order article via Infotrieve]
11. Tokime T, Nozaki K, Kikuchi H. Neuroprotective effect of FK506, an immunosuppressant, on transient global ischemia in gerbil. Neurosci Lett. 1996;206:81–84.[Medline] [Order article via Infotrieve]
12.
Nanri K, Montécot C, Springhetti V, Seylaz J,
Pinard E. The selective inhibitor of neuronal nitric oxide
synthase, 7-nitroindazole, reduces the delayed neuronal damage due to
forebrain ischemia in rats. Stroke. 1998;29:1248–1254.
13. Lei B, Adachi N, Arai T. The effect of hypothermia on H2O2 production during ischemia and reperfusion: a microdialysis study in the gerbil hippocampus. Neurosci Lett. 1997;222:91–94.[Medline] [Order article via Infotrieve]
14. Misko TP, Schilling RJ, Salvemini D, Moore WM, Currie MG. A fluorometric assay for the measurement of nitrite in biological samples. Anal Biochem. 1993;214:11–16.[Medline] [Order article via Infotrieve]
15. Marzinzig M, Nussler AK, Stadler J, Marzinzig E, Barthlen W, Nussler NC, Beger HG, Morris SM Jr, Brückner UB. Improved methods to measure end products of nitric oxide in biological fluids: nitrite, nitrate and S-nitrosothiols. Nitric Oxide. 1997;1:177–189.[Medline] [Order article via Infotrieve]
16. Spagnuolo C, Rinelli P, Coletta M, Chiancone E, Ascoli F. Oxidation reaction of human oxyhemoglobin with nitrite: a reexamination. Biochim Biophys Acta. 1987;911:59–65.[Medline] [Order article via Infotrieve]
17. Luo D, Knezevich S, Vincent SR. N-Methyl-D-aspartate-induced nitric oxide release: an in vivo microdialysis study. Neuroscience. 1993;57:897–900.[Medline] [Order article via Infotrieve]
18. Ohta K, Araki N, Shibata M, Hamada J, Komatsumoto S, Shimazu K, Fukuuchi Y. A novel in vivo assay system for consecutive measurement of brain nitric oxide production combined with the microdialysis technique. Neurosci Lett. 1994;176:165–168.[Medline] [Order article via Infotrieve]
19. Shintani F, Kanba S, Nakaki T, Sato K, Yagi G, Kato R, Asai M. Measurement by in vivo brain microdialysis of nitric oxide release in the rat cerebellum. J Psychiatr Neurosci. 1994;19:217–221.[Medline] [Order article via Infotrieve]
20. Shibata M, Araki N, Hamada J, Sasaki T, Shimazu K, Fukuuchi Y. Brain nitrite production during global ischemia and reperfusion: an in vivo microdialysis study. Brain Res. 1996;734:86–90.[Medline] [Order article via Infotrieve]
21. Togashi H, Mori K, Ueno K, Matsumoto M, Suda N, Saito H, Yoshioka M. Consecutive evaluation of nitric oxide production after transient cerebral ischemia in the rat hippocampus using in vivo brain microdialysis. Neurosci Lett. 1998;240:53–57.[Medline] [Order article via Infotrieve]
22. Malinski T, Bailey F, Zhang ZG, Chopp M. Nitric oxide measured by a porphyrinic microsensor in rat brain after transient middle cerebral artery occlusion. J Cereb Blood Flow Metab. 1993;13:355–358.[Medline] [Order article via Infotrieve]
23. Tominaga T, Sato S, Ohnishi T, Ohnishi ST. Electron paramagnetic resonance (EPR) detection of nitric oxide produced during forebrain ischemia of the rat. J Cereb Blood Flow Metab. 1994;14:715–722.[Medline] [Order article via Infotrieve]
24.
Lamontagne D, Pohl U, Busse R. Mechanical deformation
of vessel wall and shear stress determine the basal release of
endothelium-derived relaxing factor in the intact
rabbit coronary vascular bed. Circ Res. 1992;70:123–130.
25. Garthwaite J. Glutamate, nitric oxide and cell-cell signaling in the nervous system. Trends Neurosci. 1991;14:60–67.[Medline] [Order article via Infotrieve]
26. Zhang ZG, Chopp M, Gautam S, Zaloga C, Zhang RL, Schmidt HHHW, Pollock JS, Förstermann U. Upregulation of neuronal nitric oxide synthase and mRNA, and selective sparing of nitric oxide synthase-containing neurons after focal cerebral ischemia in rats. Brain Res. 1994;654:85–95.[Medline] [Order article via Infotrieve]
27.
Zhang ZG, Chopp M, Zaloga C, Pollock JS,
Förstermann U. Cerebral endothelial nitric oxide
synthase expression after focal cerebral ischemia in rats.
Stroke. 1993;24:2016–2022.
28. Lambert LE, Whitten JP, Baron BM, Cheng HC, Doherty NS, McDonald IA. Nitric oxide synthesis in the CNS, endothelium and macrophages differs in its sensitivity to inhibition by arginine analogues. Life Sci. 1991;48:69–75.[Medline] [Order article via Infotrieve]
29. Babbedge RC, Bland-Ward PA, Hart SL, Moore PK. Inhibition of rat cerebellar nitric oxide synthase by 7-nitro indazole and related substituted indazoles. Br J Pharmacol. 1993;110:225–228.[Medline] [Order article via Infotrieve]
30. Moore PK, Wallace P, Gaffen Z, Hart SL, Babbedge RC. Characterization of the novel nitric oxide synthase inhibitor 7-nitro indazole and related indazoles: antinociceptive and cardiovascular effects. Br J Pharmacol. 1993;110:219–224.[Medline] [Order article via Infotrieve]
31. Wolff DJ, Gribin BJ. The inhibition of the constitutive and inducible nitric oxide synthase isoforms by indazole agents. Arch Biochem Biophys. 1994;311:300–306.[Medline] [Order article via Infotrieve]
32. Yoshida T, Limmroth V, Irikura K, Moskowitz MA. The NOS inhibitor, 7-nitroindazole, decreases focal infarct volume but not the response to topical acetylcholine in pial vessels. J Cereb Blood Flow Metab. 1994;14:924–929.[Medline] [Order article via Infotrieve]
33.
O'Dell TJ, Huang PL, Dawson TM, Dinerman JL, Snyder
SH, Kandel ER, Fishman MC. Endothelial NOS and the
blockade of LTP by NOS inhibitors in mice lacking neuronal
NOS. Science. 1994;265:542–546.
34.
Dinerman JL, Dawson TM, Schell MJ, Snowman A, Snyder
SH. Endothelial nitric oxide synthase localized to
hippocampal pyramidal cells: implications for synaptic
plasticity. Proc Natl Acad Sci U S A. 1994;91:4214–4218.
35. Kalisch BE, Connop BP, Jhamandas K, Beninger RJ, Boegman RJ. Differential action of 7-nitro indazole on rat brain nitric oxide synthase. Neurosci Lett. 1996;219:75–78.[Medline] [Order article via Infotrieve]
36. Mackenzie GM, Rose S, Bland-Ward PA, Moore PK, Jenner P, Marsden CD. Time course of inhibition of brain nitric oxide synthase by 7-nitro indazole. Neuroreport. 1994;5:1993–1996.[Medline] [Order article via Infotrieve]
37.
Togashi H, Sasaki M, Frohman E, Taira E, Ratan RR,
Dawson TM, Dawson VL. Neuronal (type I) nitric oxide synthase regulates
nuclear factor kappaB activity and immunologic (type II) nitric oxide
synthase expression. Proc Natl Acad Sci U S A. 1997;94:2676–2680.
38.
Wendland B, Schweizer FE, Ryan TA, Nakane M, Murad F,
Scheller RH, Tsien RW. Existence of nitric oxide synthase in rat
hippocampal pyramidal cells. Proc Natl Acad Sci
U S A. 1994;91:2151–2155.
39. Schmidt-Kastner R, Freund TF. Selective vulnerability of the hippocampus in brain ischemia. Neuroscience. 1991;40:599–636.[Medline] [Order article via Infotrieve]
40. Caldwell M, O'Neill M, Earley B, Leonard B. NG-Nitro-L-arginine protects against ischemia-induced increases in nitric oxide and hippocampal neuro-degeneration in the gerbil. Eur J Pharmacol. 1994;260:191–200.[Medline] [Order article via Infotrieve]
41.
Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman
BA. Apparent hydroxyl radical production by peroxynitrite:
implications for endothelial injury from nitric oxide
and superoxide. Proc Natl Acad Sci U S A. 1990;87:1620–1624.
42.
Bechman JS, Koppenol WH. Nitric oxide, superoxide, and
peroxynitrite: the good, the bad, and the ugly. Am J
Physiol. 1996;271:C1424–C1437.
43. Shapira S, Kadar T, Weissman BA. Dose-dependent effect of nitric oxide synthase inhibition following transient forebrain ischemia in gerbils. Brain Res. 1994;668:80–84.[Medline] [Order article via Infotrieve]
Editorial Comment
Effects of 7-Nitroindazole and NG-Nitro-L-Arginine Methyl Ester
Department of Anesthesiology/Critical Care Medicine, The Johns Hopkins University, Baltimore, Maryland
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Excessive production of NO during ischemia and reperfusion is thought to contribute to neuronal injury. Studies with pharmacological inhibitors and knockout mice implicate a role of nNOS in injury from focal cerebral ischemia. An increase in brain tissue NO concentration is supported by direct measurements of NO with microsensors1 and electron paramagnetic resonance spin traps,2 and by indirect measurements with nitrite plus nitrate concentrations in jugular venous plasma.3 During global cerebral ischemia, neuroprotection by pharmacological inhibitors is less consistent, and efficacy may depend on the severity and duration of ischemia.
In the present study Lei et al systematically investigated the temporal dynamics of NO production in gerbil hippocampus as a function of ischemic duration. Nitrite plus nitrate concentration in microdialysates was used as a marker of NO production. Several interesting findings were observed.
First, in contrast to the increase in NO reported during focal ischemia, NO production appears to decrease during global ischemia. Tissue oxygenation may decrease to a greater extent in this model than in focal ischemia and may limit oxygen availability for NO formation. The large decrease in shear stress on the endothelial wall may also contribute to the decrease in NO during ischemia.
Second, NO production rapidly recovered during reperfusion after 2 or 5 minutes of ischemia. However, longer ischemic durations were required to produce an increase in NO production during reperfusion to levels above the baseline values. This excessive increase in NO was inhibited by the nNOS inhibitor 7-NI. These results are consistent with impaired recovery of calcium homeostasis after prolonged ischemia leading to stimulation of NOS in neurons.
Third, 7-NI provided a modest amount of neuroprotection in CA1 neurons when administered before 5 minutes of ischemia but not when administered after 10 minutes of ischemia. Because NO production was not elevated after 5 minutes of ischemia, the authors suggest that 7-NI may act through a nonspecific mechanism. However, it is possible that their microdialysis assay system is not adequately sensitive for detecting a short-lived burst of NO at reperfusion or that NO increases beyond the 3-hour observation period. As ischemic duration is shortened, CA1 neuronal degeneration becomes progressively delayed, and any involvement of NO might also be delayed. The lack of effect of posttreatment 7-NI after 10 minutes of ischemia could be related to the increased severity of the insult, the delayed administration of 7-NI in the 10-minute ischemia group, or persistence of activity of eNOS based in pyramidal neurons.4 Thus, some caution needs to be taken in interpreting the role of NOS in delayed neurodegeneration in hippocampus.
Fourth, 7-NI failed to decrease basal nitrite/nitrate levels in hippocampus, whereas L-NAME, which inhibits both nNOS and eNOS, reduced basal levels. These results imply that basal NO production is derived primarily from eNOS. This finding may be specific for hippocampus, where the endothelial isoform has been described in neurons.4 In other areas, 7-NI can decrease the basal level of cerebral blood flow, thereby implying tonic NO production by nNOS.5 It is also possible that the 7-NI administered in the present study does not completely inhibit nNOS. Furthermore, there may also be some leakage of nitrite/nitrate from plasma into the dialysate long after the probe insertion. L-NAME could selectively reduce this leakage by inhibiting peripheral sources of nitrite/nitrate and decreasing the diffusion gradient from plasma into brain tissue.
In summary, the present study provides new information on NOS kinetics during global ischemia and reperfusion. However, some precautions need to be considered in interpreting differences between L-NAME and 7-NI effects when applied to this indirect marker of NO production.
Received September 15, 1998; revision received December 3, 1998; accepted December 17, 1998.
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1. Malinski T, Bailey F, Zhang ZG, Chopp M. Nitric oxide measured by a porphyrinic microsensor in rat brain after transient middle cerebral artery occlusion. J Cereb Blood Flow Metab.. 1993;13:355–358.
2. Sato S, Tominaga T, Ohnishi T, Ohnishi ST. Electron paramagnetic resonance study on nitric oxide production during brain focal ischemia and reperfusion in the rat. Brain Res.. 1994;647:91–96.[Medline] [Order article via Infotrieve]
3. Kumura E, Yoshimine T, Takaoka M, Hayakawa T, Shiga T, Kosaka H. Hypothermia suppresses nitric oxide elevation during reperfusion after focal cerebral ischemia in rats. Neurosci Lett.. 1996;220:45–48.[Medline] [Order article via Infotrieve]
4. Dinerman JL, Dawson TM, Schell MJ, Snowman A, Snyder SH. Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: implications for synaptic plasticity. Proc Natl Acad Sci U S A.. 1994;91:4214–4218.
5. Kovach AGB, Lohinai Z, Marczis J, Balla I, Dawson TM, Snyder SH. The effect of hemorrhagic hypotension and retransfusion and 7-nitro-indazole on rCBF, NOS catalytic activity, and cortical NO content in the cat. Ann N Y Acad Sci.. 1994;738:348–368.[Medline] [Order article via Infotrieve]
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