(Stroke. 2000;31:2685.)
© 2000 American Heart Association, Inc.
Original Contributions |
Stroke Outcome in Double-Mutant Antioxidant Transgenic Mice
From the Departments of Anesthesiology and Critical Care Medicine (K.S., Y.A., R.J.T., P.D.H.), Neurology (A.S.M., V.L.D., T.M.D.), and Pathology (P.C.W.), Johns Hopkins University School of Medicine, Baltimore, Md.
Correspondence to Patricia D. Hurn, PhD, Departments of Anesthesiology/Critical Care Medicine, 600 N Wolfe St, Blalock 1404, The Johns Hopkins University School of Medicine, Baltimore, MD 21287-4961. E-mail phurn{at}jhmi.edu
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Abstract |
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Background and Purpose—Both NO and superoxide cytotoxicity are important in experimental stroke; however, it is unclear whether these molecules act within parallel pathological pathways or as coreagents in a common reaction. We examined these alternatives by comparing outcomes after middle cerebral artery occlusion in male and female neuronal NO synthase (nNOS)-deficient (nNOS-/-) or human CuZn superoxide dismutase–overexpressing (hSOD1+/-) mice and a novel strain with both mutations.
Methods—Permanent middle cerebral artery occlusion was performed by use of the intraluminal filament technique (18 hours). Neurological status was scored, and tissue infarction volume was determined by 2,3,5-triphenyltetrazolium staining and image analysis.
Results—Hemispheric infarction volume was reduced in each transgenic strain relative to the genetically matched, wild-type, control cohorts (WT mice): nNOS-/- (80±6 mm3) and double-mutant (49±6 mm3) mice versus WT mice (114±7 mm3) and hSOD1+/- mice (52±7 mm3) versus WT mice (95±5 mm3). Human CuZn superoxide dismutase had a larger effect on mean infarction volume (30% of contralateral hemisphere) than did nNOS deficiency (46%). Although infarction volume was less in double-mutant mice compared with nNOS-/- mice, injury was not improved relative to hSOD1+/- mice. There was no difference in histological damage by sex within each strain; however, female nNOS-/- mice were not protected from ischemic injury, unlike male mutants.
Conclusions—Superoxide generation contributes to severe ischemic brain injury in vivo to a greater extent than does neuronally derived NO. In vivo, significant superoxide scavenging by CuZn superoxide dismutase occurs within cellular compartments or through biochemical pathways that are not restricted to, and may be distinct from, neuronal NO/superoxide reaction and peroxynitrite synthesis.
Key Words: cerebral ischemia • gender • middle cerebral artery occlusion • nitric oxide synthase • stroke • superoxide dismutase • mice
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Outcome from experimental stroke is linked to a complex intertwining schema of pro-oxidant mechanisms. Both NO and superoxide anion are thought to be essential actors in oxidative injury during cerebral ischemia. Neuronal overproduction of NO has been consistently postulated to occur in the ischemic brain, leading to the death of neighboring cells. In part, NO toxicity is explained as a consequence of its propensity to react with superoxide at extremely fast rates and form the potent oxidant peroxynitrite (ONOO-). ONOO- is lipid soluble and has a wide assortment of potential oxidation targets, including proteins, RNA, and DNA.1 2 Some of the neurotoxicity of ONOO- results from depression of mitochondrial respiration3 4 and from DNA strand breakage, which stimulates energy-consuming DNA repair processes involving poly(ADP-ribose) polymerase.5 6 Pharmacological inhibitors of NO synthase (NOS) or genetic deficiency of the neuronal enzymatic isoform (nNOS) reduces NO/ONOO- toxicity and improves cell survival in many experimental paradigms (References 7 8 ; for review, see Reference 9 ). In turn, superoxide anion has long been recognized as a key oxidant species in brain injury. Numerous sources of enhanced superoxide production have been identified, including anoxic mitochondria, activated microglia and neutrophils, and membrane-bound oxidases. Therapeutic maneuvers designed to limit intraischemic increases in intracellular superoxide concentration also achieve neuroprotection (for review, see References 10 11 ). Compared with nontransgenic mice, mice overexpressing human CuZn superoxide dismutase (hSOD1) sustain less brain injury after middle cerebral artery occlusion (MCAO)12 ; extracellular superoxide dismutase (SOD)–overexpressing strains are protected in a similar manner.13 Furthermore, overexpression of manganese SOD, the mitochondrial isoform, reduces NO and ONOO- toxicity in vitro.14 15
Although it is clear that reactions involving NO or superoxide are activated in the ischemic brain, it is not known whether these molecules act within parallel pathological pathways or as coreagents in a common toxic reaction. Furthermore, it has been difficult to independently manipulate these agents in the intact brain or to determine relative contributions of NO release and superoxide generation in an animal model of neuroinjury. We hypothesized that both NO and superoxide must be present in sufficient amounts and in proximate tissue compartments if ONOO- synthesis is to be fueled. If either NO or superoxide concentration is largely ablated, then lower levels of ONOO- could result, with decreased injury to intracellular targets. Under these conditions, salvage of tissue from injury would not be different, regardless of whether NO, superoxide, or both are reduced. Alternatively, enhanced SOD1 activity through non-NO overlapping mechanisms could directly benefit the injured brain. Under these conditions, enhanced superoxide scavenging might benefit the brain in a manner additive or synergistic to the loss of neuronal NO. We used a set of transgenic mouse strains to explore these interlocking mechanisms in vivo: nNOS-deficient (nNOS-/-) mice, hSOD1 overexpressors (hSOD1+/- mice), and a novel strain in which both mutations were combined (nNOS-/-,hSOD1+/- mice). The aim was to determine whether nNOS-/-,hSOD1+/- mice exhibit improved function and smaller tissue injury after MCAO compared with mice with a single mutation.
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Materials and Methods |
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The present study was conducted in accordance with the National Institutes of Health guidelines for the care and use of animals in research and under protocols approved by the Animal Care and Use Committee of the Johns Hopkins University. Three age- and weight-matched transgenic strains were studied, and each was compared with its genetically matched wild-type strain (WT). The animal groups were as follows: (1) homozygous nNOS-null mice (nNOS-/-, founder stock16 ; n=33 total, 22 males and 11 females); (2) heterozygous hSOD1 overexpressors (hSOD+/-, founder stock17 ; n=18 total, 10 males and 8 females); (3) a novel nNOS-deficient hSOD1-overexpressing mutant strain bred on an nNOS-/- background (nNOS-/-,hSOD1+/-; n=25 total, 11 males and 14 females); (4) C57Bl/6 WT control mice for the nNOS-/- and double-mutant strains (n=20 total, 11 males and 9 females); and (5) C57Bl/6J WT control mice for the hSOD+/- strain (n=34 total, 21 males and 13 females). Because the background strain for the hSOD+/- mice was not genetically identical to that of the nNOS-/- and double-mutant mice, 2 groups of WT control mice were used. The genotype of all animals was determined by polymerase chain reaction (PCR) as described previously.16
nNOS-/- mice were produced on a purebred C57Bl/6 background as
previously described.16 18 Since their development, the
nNOS-/- mice have been continually outbred to WT C57Bl/6 (Charles
River) to avoid potential effects of strain or inbreeding. The
transgenic mice had been backcrossed to the WT mice 
8 times at the
time of the present study. Because nNOS-/- mice were therefore
>99% genetically identical to WT mice, the C57Bl/6 mice from Charles
River were used as the nNOS-/- control cohort. The
hSOD1-overexpressing mice were produced as previously described in
1995.17 These animals were originally produced in the
C57Bl/6JxHeJ hybrid strain, initially backcrossed to this same hybrid,
and then subsequently bred to the C57Bl/6J strain (Charles River). The
level of hSOD1 to endogenous mouse SOD1 activity in brain
is 8:1, with a transgene product distribution quite similar to that
of endogenous mouse enzyme.17 The hSOD1
transgenic mice have been extensively backcrossed to the C57Bl/6J WT
mice, resulting in near genetic confluence. Therefore, this WT mouse
was used in control comparisons with the hSOD1 mutant.
To develop the novel double mutants, hSOD1+/- mice were bred to WT C57/Bl6 mice, and the colony was screened by use of one PCR primer common for mouse and human SOD (5'-GTT ACA TAT AGG GGT TTA CTT CAT AAT CTG-3') and human/mouse SOD primers (5'-CAG CAG TCA CAT TGC CCA (A/GGT CTC CAA CAT G-3'). The presence of mouse SOD on PCR served as an internal PCR control, because all mice expressed mouse CuZn SOD. Female hSOD1+/- mice were then bred to nNOS -/- male mice, creating nNOS heterozygotes, some of which also overexpressed SOD (nNOS+/-,SOD+/- mice) These double heterozygotes were then bred to produce nNOS-null SOD-overexpressing mice (nNOS-/-,SOD+/- mice). This new colony was screened by using PCR primers for nNOS and SOD as described above. The PCR primers were designed to confirm the absence of nNOS (5'-CTT TCA TCT CTG CTT TGG CTG G-3', 5'-ATC TCA GAT CTG ATC CGA G-3') and the presence of neomycin (5'-CAC CAT GAT ATT CGG CAA GCA G-3', 5'-TGG AGA GGC TAT TCG GCT ATG AC-3').
Ischemic Model
Mice were anesthetized with 1% to 1.2% halothane in
oxygen-enriched air by face mask. The femoral artery was cannulated for
measurement of arterial blood gases and blood pressure.
Rectal temperature was controlled at near 37°C throughout the
experiment with heating lamps and water pads for all animals. After
baseline arterial blood gas measurement, permanent
unilateral MCAO was performed with use of an intraluminal
filament-insertion technique. The proximal common carotid artery was
ligated, and a 6-0 nylon monofilament was inserted and advanced into
the internal carotid artery to a distance of 6 mm from the
internal carotid/pterygopalatine artery bifurcation to the suture tip.
Intraischemic arterial blood pressure and blood
gases were determined after occlusion, then the catheters removed, and
anesthesia was discontinued. Neurological deficit was
confirmed in each animal at 1 hour of occlusion. Neurological deficit
was scored as follows: 0, no deficit; 1, forelimb weakness; 2, circling
to affected side; 3, unable to bear weight on affected side; and 4, no
spontaneous motor activity. If no deficit was observed, the animal was
removed from the study cohort. After 18 hours of MCAO, neurological
status was again scored, and the brain was harvested for
analysis. Infarction volume was determined by
2,3,5-triphenyltetrazolium staining in five
2-mm slices and evaluated via digital planimetry.
Statistical Analysis
All data are expressed as mean±SE.
Physiological variables and histology were
analyzed by 1-way ANOVA with a post hoc Newman-Keuls test to
correct for multiple comparisons and determine differences between
transgenic strains. Postischemic neurological scores were
analyzed by the Mann-Whitney U test. Statistical
comparisons were made between each transgenic strain and its
appropriate WT control group. Because SOD1 overexpressors were
originally developed from C57Bl/6J mice, this additional cohort was
incorporated into the study design. For simplicity, we have
presented the data analysis so that the nNOS-/- and
double-mutant mice were compared with their commonly held WT
background. Alternatively, when data from the nNOS-/- and
double-mutant mice were statistically compared with either WT cohort of
the study, the same result was obtained. Last, animals of both sexes
were used in each group. Accordingly, a post hoc analysis of
histology was carried out to determine whether there were sex
differences in infarction within each genetic group and whether the
effect of the mutation was the same in both males and females. For
this, a 1-way ANOVA with a Newman-Keuls test was used to examine total
infarction volume for each genetic group, separated by sex. A 2-way
ANOVA with a Newman-Keuls test was used to analyze effect of
the sex-matched transgenic strain (between-group comparison) on
infarction volume by brain slice (within-group comparison).
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Results |
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Arterial blood pressure, body temperature, and blood gas composition before and during MCAO remained within physiological range in all groups (Table
). In general,
intraischemic values were comparable among transgenic groups.
Figure 1 summarizes total hemispheric
infarction volume in each animal cohort, expressed as a percentage of
the contralateral hemisphere. First, each genetic modification reduced
stroke relative to the WT mouse. Infarction volume was smaller in
nNOS-/- mice (80±6 mm3) and in the
double-mutant mice (49±6 mm3) than in the
WT control mice (114±7 mm3). Similarly,
injury was reduced in hSOD1+/- mice (52±7
mm3) compared with their background WT mice
(95±5 mm3). Neurological scores at 18 hours
reflected these histological differences.
Consistent with the severity of the ischemic insult,
distinct functional deficits were observed in all animals, except for 2
females within the hSOD1+/- group, who were accorded a score of 0.
Outcome score was improved in nNOS-/- (2.5±0.2) and double-mutant
(1.9±0.1) mice compared with WT mice (3.1±0.2). Similarly, outcome
was improved in hSOD1+/- (1.6±0.2) compared with WT (3.0±0.1) mice.
Second, hSOD1 overexpression had a larger effect on infarction volume
than did nNOS deficiency. Mean infarction volume was 46% of the
contralateral hemisphere in nNOS-/- mice compared with 30% in
hSOD1+/- transgenic mice. However, neurological scores were not
different between the nNOS-/- and hSOD1 groups. Third, damage in the
double-mutant mice was not reduced relative to each single mutation.
Although infarction volume in double-mutant mice was less than that
observed in mice with the single mutation (nNOS-/-), the injury in
double-mutant mice was not different from that in hSOD1-overexpressing
mice. The neurological outcome score again paralleled improvements
in tissue injury. Although double-mutant animals scored better than did
the nNOS-/- animals after MCAO, the outcome scores were the same in
double-mutant (1.9±0.1) and hSOD1-overexpressing (1.6±0.2)
animals.
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0.05 vs respective WT strain;
+P
We also evaluated the effect of each mutation separately in male and
female animals on a post hoc basis to identify potential sex bias in
the study. There was no absolute difference in infarction between males
and females within any genetic group. However, there was one large sex
difference when the protection provided by the mutation was assessed
relative to WT control animals. Whereas male nNOS-/- animals
exhibited a clear reduction in histological damage
compared with male WT animals (Figure 2A), the female animals did not benefit
from nNOS deficiency (Figure 2B). However, the females did
benefit from hSOD1 overexpression or the double mutation. In hSOD1
overexpressors, infarction volume was reduced in both sexes relative to
their respective WT (Figure 3),
although the effect was less robust in male hSOD1+/- animals (Figure 3B). Both male and female nNOS-/-,hSOD1+/- mice sustained
equivalent total infarction volumes, and each sex was protected
compared with its respective WT (Figure 4).
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In view of a potential for sex bias in the nNOS-/- group, we repeated the analysis of infarction volume for this group when limited to only male animals. Infarction volume remained smaller in male nNOS-/- mice (76±8 mm3, n=22) compared with WT control mice (125±11 mm3).
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Discussion |
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The present study presents 2 main findings. First, double-mutant mice of both sexes that are nNOS deficient and overexpress hSOD1 sustain reduced tissue infarction relative to their genetically matched WT counterparts. However, each single mutation is not equipotent in reducing ischemic injury in vivo, because the efficacy of reducing nNOS can be increased by hSOD1 availability. Conversely, SOD1 overexpression is equally efficacious in the presence or absence of neuronal NO generation, suggesting that superoxide generation contributes more to damage than does NO toxicity after severe ischemia. Therefore, CuZn SOD may scavenge superoxide within cellular compartments or through biochemical pathways that are not restricted to, and may be distinct from, neuronal NO/superoxide reaction and ONOO- synthesis. Second, these experiments demonstrate a novel sex-based difference in stroke pathophysiology. Whereas nNOS null mice are protected in experimental stroke, as previously reported, this protection appears to be limited to the male and is not demonstrated in the female. Furthermore, sex bias in stroke outcome was not generalized to all transgenic strains in the present study. This apparent specificity could suggest that fundamental neuronal NO mechanisms of ischemic injury are redirected in the female mutant during cerebral ischemia.
The double-mutant strain was constructed to evaluate conditions in
which both neuronal NO and superoxide levels are theoretically
optimized for ischemic neuroprotection. We reasoned that if
both NO and superoxide were requisite as coreagents in ONOO- formation,
then no additional effect would be observed by a combined reduction of
both radical species. For example, a single previous study used the
nNOS inhibitor 7-nitroindazole as a means of further
reducing injury after reversible MCAO (60 minutes) in
SOD1-overexpressing mice. No additive relationship was
observed.19 Alternatively, coproduction of NO and
superoxide could enhance the toxicity of NO in an unanticipated manner,
such as through peroxide generation.20 In the present
study, we show that hSOD1 overexpression does add to the protection
offered by nNOS deficiency, nearly doubling the reduction observed in
nNOS-/- mice relative to their WT counterparts. It seems unlikely
that the quenching of ONOO- resulting from the
small residual NO generation known to occur in nNOS-/- mice could
account for this substantial reduction of infarction size.
Alternatively spliced forms of nNOS (nNOSß and
nNOS
)21 22 persist in the transgenic mice, which do not
use exon 2 for transcription (ie, the site of stop codon insertion used
to create the nNOS-/- mutant). These splice forms, particularly
nNOSß, likely account for the small amount of residual (5% to 10%)
NOS activity measured in the mice.14 23 The functional
consequence of this residual NOS activity is not known; however,
3-nitrotyrosine, a biochemical marker for ONOO-
formation, is undetectable by immunocytochemistry in the nNOS-/-
brain, even after MCAO.24 Therefore, residual neuronal NO
production in nNOS-/- mice could not account for a further
reduction of infarction in the double-mutant mice. A more reasonable
explanation is that the robust added protection provided by SOD1
overexpression on an nNOS-/- background represents important
parallel radical-scavenging mechanisms that are not tightly linked to
reduction of neuronal NO/ONOO-. One possibility
is that ONOO- synthesis is localized largely to
ischemic mitochondria, a major subcellular source of reactive
oxygen species and site of exaggerated oxidative stress. If so, then
manganese SOD may be the dominant scavenger, leaving cytoplasmic SOD1
poised to derail other sources of oxidant injury.
The benefit of the double mutation was remarkably equal to that achieved with the simple hSOD1+/- phenotype. The known mechanisms of protection by SOD are extensive, including reduced sensitivity to kainic acid–induced seizures and constitutively enhanced GABA-ergic neurotransmission,25 protection of mitochondrial respiratory function26 and blood-brain barrier integrity,27 preservation of other endogenous antioxidant moieties such as glutathione,12 and preservation of apurinic/apyrimidinic endonuclease expression and subsequent DNA fragmentation.28 Interactions with endothelial NOS (NOS 3) may be important if SOD overexpression enhances the effective half-life of NO by dismutating superoxide to hydrogen peroxide. In addition, SOD has novel actions unrelated to superoxide dismutation and can directly enhance free NO production from NOS-catalyzed L-arginine oxidation.29 If increased NO release or enhanced superoxide scavenging results in greater NO availability within the vasculature, then improved perfusion could lead to tissue salvage in the stroke penumbra. For example, treatment with a nonspecific NOS inhibitor such as NG-nitro-L-arginine methyl ester abolishes the tissue protection ordinarily observed in SOD1-overexpressing mice after MCAO.19 We did not measure cerebral blood flow in these experiments and so cannot comment on blood flow–associated mechanisms of protection. Furthermore, although the middle cerebral artery was permanently occluded in all animals, leading to a distinct neurological deficit, residual ischemic blood flow could have been different among the 3 transgenic species at any point within the 18 hours of occlusion.
The present investigation was not constructed to prospectively evaluate sex differences in the various transgenic strains. However, in view of increasing reports of sex-linked differences in outcome from neuroinjury, we studied large cohort sizes to ensure a reasonably equivalent male-to-female animal ratio. Female animals have been shown to sustain reduced stroke damage relative to males in rats,30 gerbils,31 and mice,32 and this protection is linked to female sex steroids, particularly estrogen (for review, see Reference 33 ). The present finding that female mice do not benefit from nNOS deficiency is one of the first demonstrations of a sex-based difference in a fundamental cellular mechanism of cell injury. Further experiments are needed to fully understand this observation and to determine whether the finding is replicated in reversible MCAO or other ischemic models. If estrogen (or progesterone) is implicated in this surprising result, it remains to be shown. If so, then the lack of nNOS-mediated neuroprotection in females may suggest that female sex steroids alter ischemic injury cascades at a point upstream from nNOS activation. Alternatively, estrogen and/or progesterone may protect the brain by a mechanism that parallels the genetic "blockade" of neuronal NO toxicity. Recent work indicates that female mice are also not protected by genetic deficiency in inducible NOS after permanent MCAO, unlike male mice.32 Interactions between sex steroids and NO toxicity could be quite complex because estrogen alters the expression of endothelial NOS34 35 and inducible NOS.36 37
In contrast, both male and female mice benefit from hSOD1 overexpression or the double mutation during permanent focal ischemia. These results confirm many previous reports in male mutants with global cerebral ischemia38 and with reversible,10 11 19 but not permanent, MCAO.39 There are a large number of methodological differences between the present experiments and this earlier report,39 including differing WT backgrounds (C57Bl/6J versus CD-1) and methods of transgenic strain development, duration of MCAO (18 versus 24 hours), size of occlusive monofilament (5.0 versus 6.0), and analytic methods for correction of edema. It should also be noted that the largest difference is that the study of Chan et al39 was conducted in all male animals. Even in the present study, in which a significant effect could be seen, the protection in male hSOD1 mice was not striking compared with that observed in female hSOD1 mice. It may be that native SOD1 activity is lower in female mice; hence, overexpression is highly beneficial in the female. Further studies are needed for exploration of this issue and for comparative quantification of hSOD1 transgene expression products in male versus female mutants.
In conclusion, stroke damage in a novel strain of nNOS-deficient hSOD1-overexpressing mice was equivalent to that achieved with the single hSOD1-overexpressing mutation. SOD1 overexpression reduces acute stroke damage by mechanisms not restricted to nNOS generation.
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Acknowledgments |
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This study was funded by US Public Health Service grants NS-33668, NR-03521, NS-20020, and NS-37090 (Dr T.M. Dawson) and NS-37460 (Dr V.L. Dawson). Dr T.M. Dawson is an Established Investigator of the American Heart Association. Dr V.L. Dawson is a Mary Lou McIlhany Scholar and a Staglin Music Festival NARSAD Investigator.
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Footnotes |
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Under an agreement between the Johns Hopkins University and Guilford Pharmaceuticals, Drs T.M. Dawson and V.L. Dawson are entitled to a share of sales royalty received by the University from Guilford. Dr T.M. Dawson and the University also own Guilford stock, and the University stock is subject to certain restrictions under University policy. The terms of this arrangement are being managed by the University in accordance with its conflict-of-interest policies.
Received March 27, 2000; revision received June 26, 2000; accepted July 20, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
1. Beckman JS, Chen J, Crow JP, Ye YZ. Reactions of nitric oxide, superoxide, and peroxynitrite with superoxide dismutase in neurodegeneration. Prog Brain Res.. 1994;103:371–380.[Medline] [Order article via Infotrieve]
2. Beckman JS, Chen J, Ischiropoulos H, Crow JP. Oxidative chemistry of peroxynitrite. Methods Enzymol.. 1994;233:229–240.[Medline] [Order article via Infotrieve]
3. Cassina A, Radi R. Differential inhibitory action of nitric oxide and peroxynitrite on mitochondrial electron transport. Arch Biochem Biophys. 1996;328:309–316.[Medline] [Order article via Infotrieve]
4. Yabuki M, Inai Y, Yoshioka T, Hamazaki K, Yasuda T, Inoue M, Utsumi K. Oxygen-dependent fragmentation of cellular DNA by nitric oxide. Free Radic Res. 1997;26:245–255.[Medline] [Order article via Infotrieve]
5. Eliasson MJL, Sampei K, Mandir AS, Hurn PD, Traystman RJ, Bao J, Pieper A, Wang ZQ, Dawson TM, Snyder SH, Dawson VL. Poly (ADP-ribose) polymerase gene disruption renders mice resistant to cerebral ischemia. Nat Med. 1997;3:1089–1095.[Medline] [Order article via Infotrieve]
6. Endres M, Wang ZQ, Namura S, Waeber C, Moskowitz MA. Ischemic brain injury is mediated by the activation of poly (ADP-ribose) polymerase. J Cereb Blood Flow Metab. 1997;17:1143–1151.[Medline] [Order article via Infotrieve]
7. Huang Z, Huang PL, Panahian N, Dalkara T, Fishman MC, Moskowitz MA. Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science. 1994;265:1882–1885.
8.
Dawson VL, Kizushi VM, Huang PL, Snyder SH, Dawson TM.
Resistance to neurotoxicity in cortical cultures from neuronal nitric
oxide synthase-deficient mice. J Neurosci. 1996;16:2479–2487.
9. Dawson VL, Dawson TM. Nitric oxide in neurodegeneration. Prog Brain Res. 1998;118:215–228.[Medline] [Order article via Infotrieve]
10. Kinouchi H; Kamii H; Mikawa S; Epstein CJ; Yoshimoto T; Chan PH. Role of superoxide dismutase in ischemic brain injury. Cell Mol Neurobiol. 1998;18:609–620.[Medline] [Order article via Infotrieve]
11.
Chan PH. Role of oxidants in ischemic brain
damage. Stroke. 1996;27:1124–1129.
12.
Kinouchi H, Epstein CJ, Mizui T, Carlson E, Chen SF,
Chan PH. Attenuation of focal cerebral ischemic injury in
transgenic mice overexpressing CuZn dismutase. Proc Natl Acad Sci
U S A. 1991;88:11158–11162.
13. Sheng H, Bart RD, Oury TD, Pearlstein RD, Crapo JD, Warner DS. Mice overexpressing extracellular superoxide dismutase have increased resistance to focal cerebral ischemia. Neuroscience. 1999;88:185–191.[Medline] [Order article via Infotrieve]
14.
Gonzalez-Zulueta M, Ensz LM, Mukhina G, Lebovitz RM,
Zwacka RM, Engelhardt JF, Oberley LW, Dawson VL, Dawson TM. Manganese
superoxide dismutase protects nNOS neurons from NMDA and nitric
oxide-mediated neurotoxicity. J Neurosci. 1998;18:2040–2055.
15.
Keller JN, Kindy MS, Holtsberg FW, St Clair DK, Yen HC,
Germeyer A, Steiner SM, Bruce-Keller AJ, Hutchins JB, Mattson MP.
Mitochondrial manganese superoxide dismutase prevents neural
apoptosis and reduces ischemic brain injury:
suppression of peroxynitrite production, lipid peroxidation and
mitochondrial dysfunction. J Neurosci. 1998;18:687–697.
16. Huang PL, Dawson TM, Bredt DS, Snyder SH, Fishman MC. Targeted disruption of the neuronal nitric oxide synthase gene. Cell. 1993;75:1273–1286.[Medline] [Order article via Infotrieve]
17. Wong PC, Pardo CA, Borchelt DR, Lee MK, Copeland NG, Jenkins NA, Sisodia SS, Cleveland DW, Price DL. An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria. Neuron. 1995;14:1105–1116.[Medline] [Order article via Infotrieve]
18.
Przedborski S, Jackson-Lewis V, Yokoyama R, Shibata T,
Dawson VL, Dawson TM. Role of neuronal nitric oxide in
1-methyl-4-phenyl-1,2,3,6-tetradydropyridine (MPTP)-induced
dopaminergic neurotoxicity. Proc Natl Acad Sci U S A. 1996;93:4565–4571.
19. Kamii H, Mikawa S, Murakami K, Kinouchi H, Yoshimoto T, Reola L, Carlson E, Epstein CJ, Chan PH. Effects of nitric oxide synthase inhibition on brain infarction in SOD-1-transgenic mice following transient focal cerebral ischemia. J Cereb Blood Flow Metab. 1996;16:1153–1157.[Medline] [Order article via Infotrieve]
20.
Gobbel GT, Chan TY, Chan PH. Nitric oxide and
superoxide-mediated toxicity in cerebral endothelial
cells. J Pharmacol Exp Ther. 1997;282:1600–1607.
21.
Brenman JE, Chao DS, Gee SH, McGee AW, Craven SE,
Santillano DR, Uw Z, Huang F, Xia H, Peters MF, Froehner SC, Bredt DS.
Interaction of nitric oxide synthase with the postsynaptic density
protein PSD-95 and 
1-syntrophin mediated by PDZ domains.
Cell. 1996;84:757–767.[Medline]
[Order article via Infotrieve]
22.
Eliasson MJL, Blackshaw S, Schell MJ, Snyder SH.
Neuronal nitric oxide synthase alternatively spiced forms: prominent
functional localizations in the brain. Proc Natl Acad Sci
U S A. 1997;94:3396–3401.
23. Wei G, Dawson VL, Zweier JL. Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia. Biochim Biophys Acta. 1999;1455:23–34.[Medline] [Order article via Infotrieve]
24.
Eliasson MJL, Huang Z, Ferrante RJ, Sasamata M,
Molliver ME, Snyder SH, Moskowitz MA. Neuronal nitric oxide synthase
activation and peroxynitrite formation in ischemic stroke
linked to neural damage. J Neurosci. 1999;19:5910–5918.
25.
Levkovitz Y, Avignone E, Groner Y, Segal M.
Upregulation of GABA neurotransmission suppresses hippocampal
excitability and prevents long-term potentiation in transgenic
superoxide dismutase-overexpressing mice. J Neurosci. 1999;19:10977–10984.
26. Merad-Saidoune M, Boitier E, Nicole A, Marsac C, Martinous JC, Sola B, Sinet PM, Ceballos-Picot I. Overproduction of Cu-Zn-superoxide dismutase or Bcl-2 prevents the brain mitochondrial respiratory dysfunction induced by glutathione depletion. Exp Neurol. 1999;158:428–436.[Medline] [Order article via Infotrieve]
27. Kondo T, Reaume AG, Huang TT, Carlson E, Murakami K, Chen SF, Hoffman ED, Scott RW, Epstein CJ, Chan PH. Reduction of Cu-Zn-superoxide dismutase activity exacerbates neuronal cell injury and edema formation after transient focal cerebral ischemia. J Neurosci. 1997;11:4180–4189.
28.
Fujimura M, Morita-Fujimura Y, Narasimhan P, Copin JC,
Kawase M, Chan PH. Copper-zinc superoxide dismutase prevents the early
decrease of apurinic/apyrimidinic endonuclease and subsequent DNA
fragmentation after transient focal cerebral ischemia in mice.
Stroke. 1999;30:2408–2415.
29.
Hobbs AJ, Fukuto JM, Ignarro LJ. Formation of free
nitric oxide from L-arginine by nitric oxide synthase: direct
enhancement of generation by superoxide dismutase. Proc Natl Acad
Sci U S A. 1994;91:10992–10996.
30.
Alkayed NJ, Harukuni I, Kimes AS, London ED, Traystman
RJ, Hurn PD. Gender-linked brain injury in experimental stroke.
Stroke. 1998;29:159–165.
31. Hall ED, Pazara KE, Linseman KL. Sex differences in postischemic neuronal necrosis in gerbils. J Cereb Blood Flow Metab. 1991;11:292–298.[Medline] [Order article via Infotrieve]
32. Loihl AK, Asensio V, Campbell IL, Murphy S. Expression of nitric oxide synthase (NOS)-2 following permanent focal ischemia and the role of nitric oxide in infarct generation in male, female, and NOS-2 gene-deficient mice. Brain Res. 1999;830:155–164.[Medline] [Order article via Infotrieve]
33. Hurn PD, Macrae IM. Estrogen as neuroprotectant in stroke. J Cereb Blood Flow Metab. 2000;20:631–652.[Medline] [Order article via Infotrieve]
34.
Goetz RM, Thatte HS, Prabhakar P, Cho MR, Michel T,
Golan DE. Estradiol induces the calcium-dependent translocation of
endothelial nitric oxide synthase. Proc Natl Acad
Sci U S A. 1999;96:2788–2793.
35.
Kleinert H, Wallerath T, Euchenhofer C, Ihrig-Biedert
I, Li H, Forstermann U. Estrogens increase transcription of the
human endothelial NO synthase gene: analysis of
the transcription factors involved. Hypertension. 1998;31:582–588.
36. Miller L, Alley EW, Murphy W, Russell SW, Hunt JS. Progesterone inhibits inducible nitric oxide synthase gene expression and nitric oxide production in murine macrophages. J Leukoc Biol. 1996;59:442–450.[Abstract]
37. Hayashi T, Yamada K, Esaki T, Muto E, Chaudhuri G, Iguchi A. Physiological concentrations of 17ß estradiol inhibit the synthesis of nitric oxide synthase in macrophages via a receptor-mediated system. J Cardiovasc Pharmacol. 1998;31:292–298.[Medline] [Order article via Infotrieve]
38.
Chan PH, Kawase M, Murakami K, Chen SF, Li Y, Calagui
B, Reola L, Carlson E, Epstein CJ. Overexpression of SOD1 in transgenic
rats protects vulnerable neurons against ischemic damage after
global cerebral ischemia and reperfusion. J
Neurosci. 1998;18:8292–8299.
39. Chan PH, Kamii H, Yang G, Gafni J, Epstein CJ, Carlson E, Reola L. Brain infarction is not reduced in SOD-1 transgenic mice after a permanent focal cerebral ischemia. Neuroreport. 1993;5:293–296.[Medline] [Order article via Infotrieve]
Editorial Comment
Department of Neurology Washington University School of Medicine St Louis, Missouri
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Both superoxide anion and nitric oxide (NO) have been implicated in the pathogenesis of ischemic brain injury. Genetically engineered mice have been used to delineate the function of single genes that alter superoxide anion or neuronal NO formation in cerebral ischemia. Thus, mice overexpressing CuZn superoxide dismutase (SOD) with enhanced capacity to scavenge superoxide anion were more resistant to ischemic insult.R1 Similarly, mice deficient in neuronal nitric oxide synthase (nNOS) sustained lesser degrees of ischemic brain injury.R2 Superoxide anion and NO interact to form peroxynitrite, a highly reactive and toxic free radical species that causes tissue damage.R3 Sampei and coworkers applied a novel mouse strain harboring double mutations characterized by CuZn SOD overexpression and nNOS deletion to explore possible synergistic or additive neuroprotective effects of reducing superoxide anion and neuronal NO generation. An important observation in the present study is the comparison of the effectiveness of the 2 types of mutations, namely, nNOS deletion versus CuZn SOD overexpression, in mice with otherwise identical genetic backgrounds. This study is the first to show CuZn SOD overexpression to be more potent than nNOS deletion in protecting the brain from ischemia. The findings are also interesting in that mice with double mutations were better protected than mice deficient in nNOS. In contrast, CuZn SOD overexpression alone was as effective as double mutations in reducing ischemic brain injury. These results strongly suggest a broader action of superoxide anion, beyond its interaction with NO to form peroxynitrite, in the pathogenesis of ischemic brain injury.
The senior author of the present study, Dr Hurn, is a leading authority in gender differences in brain vulnerability to ischemic insult.R4 It is interesting to note a gender effect of nNOS deletion such that male but not female mice benefited from nNOS deletion. A possible gender difference is also noted in mice overexpressing CuZn SOD. The gender effects disclosed by gene manipulations provide important insights into the interactions of female hormones with ischemia-induced brain injury cascades.
Received March 27, 2000; revision received June 26, 2000; accepted July 20, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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1. Kinouchi H, Epstein CJ, Mizui T, Carlson E, Cgeb SF, Chan PH. Attenuation of focal cerebral ischemic injury in transgenic mice overexpressing CuZn superoxide dismutase. Proc Natl Acad Sci U S A.. 1991;88:11158–11162.
2. Huang Z, Huang PL, Panahian N, Dalkara T, Fishman MC, Moskowitz MA. Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science.. 1994;265:1882–1885.
3.
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.
4. Hurn PD, Macrae IM. Estrogen as neuroprotectant in stroke. J Cereb Blood Flow Metab.. 2000;20:631–652.
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