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(Stroke. 2000;31:2208.)
© 2000 American Heart Association, Inc.
Original Contributions |
Subacute But Not Acute Generation of Nitric Oxide in Focal Cerebral Ischemia
From the Department of Neurology (K.F., M.F., A.R., S.K., M.H.) and Institute of Clinical Chemistry (T.B.), University of Heidelberg, Mannheim; and Center of Molecular Biology, ZMBH, University of Heidelberg, Heidelberg (M.P.), Germany.
Correspondence to PD Dr K. Fassbender, Department of Neurology, University of Heidelberg at Mannheim, Theodor-Kutzer-Ufer 1-3, 68135 Mannheim, Germany.
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Abstract |
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Background and Purpose—Excessive release of nitric oxide (NO) has been implicated in the pathophysiology of neurodegeneration in ischemic stroke. We compared intracerebral release of indicators of NO generation at the acute and subacute stages of transient focal cerebral ischemia.
Methods—In vivo microdialysis in the rat striatum was performed at the acute (first hours) and subacute (after 24 or 48 hours) stages of cerebral ischemia or sham operation to monitor intracerebral release of the stable NO metabolites nitrite and nitrate.
Results—Whereas only a nonsignificant trend toward increased release of these NO metabolites was evidenced in acute cerebral ischemia, a significant NO generation was observed subacutely, 48 hours after induction of cerebral ischemia. Aminoguanidine, a selective inhibitor of inducible NO synthase, suppressed this delayed release of nitrite and nitrate.
Conclusions—Whereas these observations do not support a major NO generation in acute cerebral ischemia, they indicate an inducible NO synthase–dependent NO generation predominantly at the subacute phase of ischemic neurodegeneration. Therefore, NO generation may play a pathophysiological role in delayed ischemic neurodegeneration.
Key Words: cerebral ischemia, focal • nitric oxide • nitric oxide synthase • rats
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Whereas in low concentrations (nanomolar range), nitric oxide (NO) plays a physiological role in neuronal signaling, in high concentrations (micromolar range) this molecule is enormously cytotoxic.1 The production of NO, itself a free radical, promotes tissue injury, eg, by reaction with superoxide anion to produce the extremely toxic peroxynitrite or by interaction with proteins, transition metals, and iron-sulfur–containing or heme-containing compounds.2 3 Excessive NO generation has been implicated in ischemic neurodegeneration4 5 and is currently intensely studied as a therapeutic target in ischemic stroke.5 6
The rapidly decaying gas NO cannot be detected directly. However, changes in concentrations of the stable NO oxidation products nitrite and nitrate allow estimation of NO generation.7 Recently, extracellular release of NO metabolites in the central nervous system has been reported in acute global8 9 10 11 or focal12 13 cerebral ischemia with the use of in vivo microdialysis. This technique allows repeated sampling of brain tissue perfusate in the same freely moving rat.14 The results of these studies were conflicting since they showed decreased or increased concentrations of NO metabolites for minutes or hours. More importantly, whereas formation of NO metabolites has been monitored with this technique only in the acute stage of focal cerebral ischemia, there is, to our knowledge, no information about a possible NO generation in the subacute phase (ie, beyond 24 hours). This uncertainty clearly contrasts with the general belief that NO is a key molecule in the pathogenesis of ischemic neurodegeneration and represents an important therapeutic target in ischemic stroke.5 In this study we comparatively studied the intracerebral generation of NO at the acute and subacute stages of transient focal cerebral ischemia.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stroke Models
Male adult albino rats of a Wistar-derived strain (Charles River, Wiga, Germany) weighing 240 to 440 g, were housed under standard conditions with free access to food and water. Infarcts were induced as previously described.15 16 Briefly, as the rats were monitored for blood pressure, temperature, and oxygenation, the right carotid artery was exposed after median incision of the neck skin. The right middle cerebral artery (MCA) was occluded with a silicone rubber cylinder attached to a nylon surgical thread introduced from the common carotid artery immediately after ligation of the ipsilateral external and proximal common carotid artery. The cylinder was made of a 4-0 nylon surgical thread, coated with silicone and hardener to thicken the distal 5 mm to 0.25 to 0.30 mm. The thread was advanced for 16 to 20 mm into the lumen of the internal carotid artery until it blocked the origin of the MCA. Then the internal carotid artery was ligated just distal to the point of insertion. Recirculation was performed by pulling the thread out after 2 hours. The sham operation was performed as described above, except that the filament was not advanced to block the MCA.
In Vivo Microdialysis
Under conditions similar to those of the in vivo experiments
(perfusion rate of 2 µL/min, 37°C), an in vitro recovery for
nitrite dissolved in Ringer’s solution of 33±4% was obtained. In
vivo microdialysis was performed as previously
described.17 Rats were anesthetized with a mixture
of 2% fentanyl (6 mg/kg body wt IP) plus medetomidin (0.3 mg/kg body
wt IP) and placed in a stereotaxic apparatus.
Thereafter, the dura was exposed through a 2-mm hole in the skull, and
the microdialysis probe was inserted in the striatum. The coordinates
to which the tip of the dialysis probe (100 000-Da cutoff, CMA
12) was lowered with respect to bregma and dural surfaces were
as follows: anterior +1.7 mm, lateral -2.0 mm, and vertical
6.0 mm, with the incisor bar at -2.5 mm.18
After implantation, the probe was secured with dental cement to
2 small anchoring screws on the top of the skull. After surgery, the
animals were housed with free access to food and water. A modified
Ringer’s solution consisting of (mmol/L) NaCl 121, KCl 3.5,
MgCl2 1.2, CaCl2 1.2,
NaH2PO4 1.0,
NaHCO3 25 (pH 7.4) was constantly perfused
through the implanted probe at a rate of 2 µL/min with the use of a
microinjection pump. Dialysates were collected every 10 minutes and
immediately frozen.
Experimental Conditions
In each experimental group 6 to 8 animals were studied. For
analysis of NO generation in acute cerebral ischemia,
microdialysis probes were implanted 24 hours before the induction of
cerebral ischemia or sham operation. Microdialysis was started
3 hours before ischemia to equilibrate the system for the first
hour and to obtain baseline values for the 2 subsequent hours. In this
group measurements were performed directly before and after induction
of experimental ischemia and reperfusion (or sham
operation).
For determination of NO generation in subacute cerebral ischemia, microdialysis was performed beginning either 24 or 48 hours after induction of ischemia. Under these conditions, microdialysis probes were implanted and perfused 3 hours before the first measurements to equilibrate the system. In an additional study group, the effects of the selective inhibitor of inducible NO synthase (iNOS), aminoguanidine, on subacute NO generation were tested, at a dosage previously described (400 mg/kg IP, twice daily, beginning on the day of experimental ischemia).19
Analysis of Release of NO Metabolites
NO was quantified in microdialysates via the nitrite method
based on the Griess reaction, with the use of a
colorimetric assay from Boehringer. Dialysates
collected within intervals of 1 hour were pooled to obtain 2 fractions
for duplicate determinations. The nitrate present in the sample is
reduced to nitrite by reduced nicotinamide adenine
dinucleotide phosphate in the presence of the nitrate
reductase. The nitrite formed reacts with sulfanilamide and
N-(1-naphthyl)-ethylenediamine dihydrochloride to yield
a red-violet diazo dye, which is measured on the basis of its
absorbance in the range of 550 nm. Known concentrations of sodium
nitrite were included as standards. The lower limit of detection is
0.32 µmol/L for nitrite. The intratest variance is <10%, and
the intertest variance is <20%.
Statistical Analysis
Results are expressed as mean (±SE). The Mann-Whitney test was
used for the nonparametric statistical analysis.
Differences were considered significant at P<0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hemodynamic Variables, Changes in Body Weight, and Neurological Scores
Hemodynamic variables were within the same range in animals with ischemia and sham operation. Thus, mean arterial pressures during the operation were 115.52±16.50 and 101.43±11.54 mm Hg in the sham-operated groups, 113.15±6.79 mm Hg in the group with acute ischemia, and 127.26±21.01 and 101.72±10.97 mm Hg in the groups with subacute ischemia. Repeated measurements of body weight revealed a slight weight loss 2 days after surgery in both sham-operated (from 324.3±27.1 to 321.3±24.2 g) and ischemic (from 330.8±37.3 to 308.3±26.3 g) animals. This decrease in body weights did not significantly differ between both groups. Neurological scores according to Bederson et al20 tended to be slightly better in animals that had experimental ischemia 48 hours before (2.3±1.9) compared with those that had ischemia 24 hours before (3.5±1.0).
NO Generation in Acute Cerebral Ischemia
In sham-operated animals, extracellular release of NO metabolites
was detectable that was stable in time (Figures 1 to 3
).
However, compared with these control animals, no significant changes in
nitrite/nitrate release were observed in animals with acute cerebral
ischemia (Figure 1). We observed only a minor,
nonsignificant trend toward increased release of the NO metabolites
peaking directly after induction of ischemia and, again,
immediately after reperfusion (Figure 1).
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NO Generation in Subacute Cerebral Ischemia and its
Suppression by Aminoguanidine
Twenty-four hours after onset of cerebral ischemia,
concentrations of nitrite/nitrate in dialysates tended to be
transiently increased, although this increase was not significant
compared with sham-operated animals (Figure 2
). However, 48
hours after induction of experimental cerebral ischemia,
extracellular concentrations of NO metabolites were significantly
increased (Figure 3). Levels peaked after 2 hours of measurement
(6 hours after initiation of microdialysis) and declined thereafter.
Administration of the iNOS inhibitor aminoguanidine
completely inhibited this pronounced delayed NO generation (Figure 3).
Histological Analyses
Histological examination (hematoxylin-eosin
staining of coronary 10-µm brain sections) confirmed
infarctions in the MCA territory, their lack in the sham-operated
group, and the correct placement of the microdialysis probes.
Percentages of the infarcted area compared with the total hemisphere
were 77±5% (quantified at the coronary planes in the area of
the tip of the dialysis probes with a computer-assisted planimetry
device). Using perfusion with ink, we confirmed in additional animals
that no reperfusion occurred in permanent MCA occlusion (filament not
drawn), whereas nearly the complete MCA territory was reperfused after
transient MCA occlusion for 2 hours except for a few small spots
(no-reflow phenomenon21 ).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite the immense current interest in the role of NO generation as a therapeutic target in ischemic stroke, in vivo data on release of this molecule in cerebral infarction are still scarce. In this study we report that NO metabolites are released predominantly in subacute cerebral ischemia and only in minute amounts in acute disease.
Our method was sensitive enough to detect a release of NO metabolites even in nonischemic brain (sham operation) and under basal conditions before induction of ischemia in quantities reported in earlier studies.8 12 These basal nitrite levels were extremely stable as a precondition to detect even minor changes in concentrations. Dialysis probes were intracerebrally placed at different intervals in animals with acute and subacute infarction to avoid prolonged insertion times of the microdialysis probes beyond limits that might have caused nonspecific local inflammation (and iNOS expression). We studied changes in relation to sham-operated control groups, controlling for such methodological problems.
Although we observed a nonsignificant trend toward transiently increased concentrations during ischemia and again immediately after reperfusion, these changes were minute compared with the NO generation observed in subacute disease. This delayed release of NO metabolites in the high, cytotoxic concentration range is the major finding of this study. Whereas a trend toward increased nitrite release was detected 24 hours after induction of cerebral ischemia, nitrite release was significantly increased at day 2. Interestingly, such predominantly delayed NO generation is in accordance with the recent detection of NO metabolites in brain homogenates predominantly 3 days after transient cerebral ischemia22 and agrees exactly with the observation of subacute glial activation and intracerebral migration of macrophages after 2 days at sites of ischemic injury.23 24 These cells harbor the subacutely synthesized iNOS19 22 that mediates generation of much larger quantities of NO25 than the constitutive isoenzymes held responsible for NO production in acute stroke.26 In such an environment, further cerebral injury associated with insertion of the dialysis probes could trigger a maximal iNOS-dependent NO generation. Indeed, the inhibitory effects of aminoguanidine delineate such a crucial role of iNOS expression in subacute cerebral ischemia. A beneficial effect of aminoguanidine on resulting infarct volumes has already been shown in earlier works,19 27 28 and its confirmatory evaluation was beyond the aims of this longitudinal study.
The transient temporal profile of NO release in subacute cerebral ischemia is unclear. It is unlikely that dietary differences could explain increased NO generation in cerebral ischemia since postoperative changes in body weights did not significantly differ between ischemic and sham-operated groups.
This, to our knowledge, first study comparing NO generation at different stages of focal cerebral ischemia supports a pathophysiological role of iNOS-dependent NO generation predominantly in the subacute phase of disease. It should be noted that the results of this study need to be considered in relation to the chosen animal model.
This predominantly subacute NO generation could explain the large number of negative studies investigating the therapeutic effects of nonspecific or constitutive NOS inhibitors in acute cerebral ischemia.6 29 30 31 At the same time, this study reveals a high susceptibility of ischemically injured brain tissue to respond to further noxious stimuli (eg, infection, blood pressure variations, or dehydration) by excessive NO generation. This raises the possibility that interventions in NO-mediated toxicity could be neuroprotective even 1 to 2 days after onset of cerebral ischemia, as suggested in a recent preliminary report.27
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Acknowledgments |
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We are grateful to Dr W. Kuschinsky for critical reading of the manuscript and M. Garcia-Knapp for secretarial assistance in the preparation of the manuscript.
Received March 10, 2000; revision received June 15, 2000; accepted June 19, 2000.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Dawson V, Brahmbhatt HP, Mong JA, Dawson TM. Expression of inducible nitric oxide synthase causes delayed neurotoxicity in primary mixed neuronal-glial cortical cultures. Neuropharmacology. 1994;33:1425–1430.[Medline] [Order article via Infotrieve]
2.
Beckman JS, Beckman TW, Chen TW, Marshall PA, Freeman
BA. Apparent hydroxy radical production by peroxynitrite:
implications for endothelial cell injury from nitric
oxide and superoxide. Proc Natl Acad Sci U S A. 1990;87:1620–1624.
3. Stamler JS. Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell. 1994;78:931–936.[Medline] [Order article via Infotrieve]
4. Iadecola C. Bright and dark sides of nitric oxide in ischemic brain damage. Trends Neurosci. 1997;20:132–138.[Medline] [Order article via Infotrieve]
5.
O‘Mahony D, Kendall MJ. Nitric oxide in acute
ischaemic stroke: a target for neuroprotection. J Neurol
Neurosurg Psychiatry. 1999;67:1–3.
6. Wahlgren N. Neuroprotectants in late clinical development: a status report. Cerebrovasc Dis. 1997;7(suppl 2):13–17.
7. Dawson TM, Steiner JP, Dawson VL, Dinerman JL, Uhl GR, Snyder SH. Immunosuppressant FK506 enhances phosphorylation of nitric oxide synthase and protects against glutamate neurotoxicity. Proc Natl Acad Sci U S A. 1993;90:9809–9812.
8. 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]
9. Sugimura T, Sako K, Tohyama Y, Yonemasu Y. Consecutive in vivo measurement of nitric oxide in transient forebrain ischemic brain under normothermia and hypothermia. Brain Res. 1998;808:313–316.[Medline] [Order article via Infotrieve]
10. 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]
11.
Lei B, Adachi N, Nagaro T, Arai T, Koehler RC. 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. Stroke. 1999;30:669–677.
12.
Kader A, Frazzini VI, Solomon RA, Trifiletti RR. Nitric
oxide production during focal cerebral ischemia in
rats. Stroke. 1993;24:1709–1716.
13.
Toung TJ, Bhardwaj A, Dawson VL, Dawson TM, Traystman
RJ, Hurn PD. Neuroprotective FK506 does not alter in vivo nitric oxide
production during ischemia and early reperfusion in
rats. Stroke. 1999;30:1279–1285.
14. Benviste H. Brain microdialysis. J Neurochem. 1989;52:1667–1679.[Medline] [Order article via Infotrieve]
15.
Nagasawa H, Kogure K. Correlation between cerebral
blood flow and histologic changes in a new rat model of middle cerebral
artery occlusion, Stroke.. 1989;20:1037–1043.
16.
Zea-Longa E, Weinstein PR, Carlson S. Reversible middle
cerebral artery occlusion without craniectomy in rats.
Stroke. 1989;20:84–81.
17.
Ishii K, Muelhauser F, Liebl U, Picard M, Penke B,
Bayer T, Hennerici M, Hartmann T, Beyreuther K, Fassbender K.
Subacute NO generation induced by Alzheimer’s
ß-amyloid in the living brain: reversal by inhibition of the
inducible NO synthase. FASEB J. 2000;14:1485–1489.
18. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. Sydney, Australia: Academic Press; 1982.
19.
Iadecola C, Zhang F, Xu X. Inhibition of inducible
nitric oxide synthase ameliorates cerebral ischemic damage.
Am J Physiol. 1995;268:R286–R292.
20.
Bederson JB, Pitts LH, Tsui M, Nishimura MC, Davies RL,
Bartkowski H. Rat middle cerebral artery occlusion: evaluation of the
model and development of a neurologic examination. Stroke. 1986;17:472–476.
21. Ames A, Wright LW, Kowade M, Thurston JM, Majors G. Cerebral ischemia, II: the no-reflow phenomenon. Am J Pathol. 1968;52:437–453.[Medline] [Order article via Infotrieve]
22. Grandati M, Verrecchia C, Revaud ML, Allix M, Boulu RG, Plotkine M. Calcium-independent NO-synthase activity and nitrites/nitrates production in transient focal ischaemia in mice. Br J Pharmacol. 1997;122:625–630.[Medline] [Order article via Infotrieve]
23.
Kochanek PM, Hallenbeck JM. Polymorphonuclear
leukocytes and monocytes/macro-phages in the pathogenesis of cerebral
ischemia and stroke. Stroke. 1992;23:1367–1379.
24. Garcia JH, Liu KF, Yoshida Y, Lian J, Chen S, Del Zoppo GJ. Influx of leukocytes and platelets in an evolving brain infarct (Wistar rat). Am J Pathol. 1994;44:188–199.
25.
Stuehr DJ, Cho HJ, Kwon NS, Weise MF, Nathan CF.
Purification and characterization of the cytokine-induced
macrophage nitric oxide synthase: an FAD- and FMN-containing
flavoprotein. Proc Natl Acad Sci U S A. 1991;88:7773–7777.
26. Zhang ZG, Chopp M, Gautam S, Zaloga RL, Schmidt HHW. Upregulation of neuronal nitric oxide synthase and mRNA, and selective sparing of nitric oxide synthase-containing neurons after focal cerebral ischemia in rat. Brain Res. 1994;654:85–95.[Medline] [Order article via Infotrieve]
27. Nagayama M, Zhang F, Iadecola C. Post-treatment with aminoguanidine reduces cerebral ischemic damage and neurological deficits in rats with middle cerebral artery occlusion. Stroke. 1998;29:331. Abstract.
28.
Zhang F, Casey RM, Ross ME, Iadecola C. Aminoguanidine
ameliorates and L-arginine worsens brain damage from
intraluminal middle cerebral artery occlusion. Stroke. 1996;27:317–323.
29. Yamamoto S, Golanov EV, Berger SB, Reis DJ. Inhibition of nitric oxide synthesis increases focal ischemic infarction in rat. J Cereb Blood Flow Metab.. 1992;12:717–726.[Medline] [Order article via Infotrieve]
30.
Kirsch JR, Bhardwaj 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.
31. Dawson DA, Kusumoto K, Graham DI, McCulloch J, Macrae IM. Inhibition of nitric oxide synthase does not reduce infarct volume in a rat model of focal cerebral ischemia. Neurosci Lett. 1992;142:151–154.[Medline] [Order article via Infotrieve]
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