(Stroke. 1999;30:1962-1968.)
© 1999 American Heart Association, Inc.
Original Contributions |
Exacerbation of Delayed Cell Injury After Transient Global Ischemia in Mutant Mice With CuZn Superoxide Dismutase Deficiency
From the Departments of Neurosurgery, Neurology and Neurological Sciences and the Program in Neurosciences, Stanford University School of Medicine, Palo Alto, Calif (M.K., M.F., Y.M-F., Y.G., P.H.C.); Departments of Neurological Surgery and Neurology, CNS Injury and Edema Research Center, University of California, San Francisco (M.K., K.M., M.F., Y.M-F., T.K., P.H.C.); and Department of Molecular Biology, Cephalon, Inc, West Chester, Pa (R.W.S.).
Correspondence to Pak H. Chan, PhD, Neurosurgical Labs, Stanford University, 701B Welch Rd, #148, Palo Alto, CA 94304. E-mail phchan{at}leland.stanford.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
|---|
Background and Purpose—We have demonstrated that copper-zinc superoxide dismutase (CuZn-SOD), a cytosolic isoenzyme of SODs, has a protective role in the pathogenesis of superoxide radical–mediated brain injury. Using mice bearing a disruption of the CuZn-SOD gene (Sod1), the present study was designed to clarify the role of superoxide anion in the pathogenesis of selective vulnerability after transient global ischemia.
Methods—Sod1 knockout homozygous mutant mice (Sod1 -/-) with a complete absence of endogenous CuZn-SOD activity, heterozygous mutant mice (Sod1 +/-) with a 50% decrease in the activity, and littermate wild-type mice (male, 35 to 45 g) were subjected to global ischemia. Since the plasticity of the posterior communicating artery (PcomA) has been reported to influence the outcome of hippocampal injury, we assessed the relation between the plasticity of PcomAs and the decrease of regional cerebral blood flow in global ischemia.
Results—The fluorescence intensity of hydroethidine oxidation, a measurement of ethidium fluorescence for superoxide radicals, was increased in mutant mice 1 day after both 5 and 10 minutes of global ischemia, compared with wild-type mice. Hippocampal injury in the PcomA hypoplastic brains showed significant exacerbation in mutant mice compared with wild-type littermates 3 days after 5 minutes of global ischemia, although a marked difference was not observed at 1 day.
Conclusions—These data suggest that superoxide radicals play an important role in the pathogenesis of delayed injury in the vulnerable hippocampal CA1 subregion after transient global ischemia.
Key Words: cerebral ischemia, global • free radicals • hippocampus • superoxide dismutase • superoxides • oxidative stress • mice
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
|---|
Oxygen radicals have been implicated in the pathogenesis of many neurological disorders and ischemic neuronal injury.1 2 3 One role of oxygen radicals appears to involve reperfusion after cerebral ischemia.4 Reperfusion supplies oxygen to the ischemic region, but oxygen could be a substrate for oxidative reaction producing oxygen radicals. Overproduced oxygen radicals that exceed the capacity of the endogenous antioxidant enzymes, including superoxide dismutase (SOD), glutathione peroxidase, and catalase, cause oxidative stress or injury in cerebral cells. Among these oxygen radicals, superoxide anions (O2-), being directly toxic to neurons,5 6 may initiate a free radical–mediated chain reaction causing additional central nervous system damage.3 We have demonstrated that copper-zinc SOD (CuZn-SOD), a cytosolic isoenzyme of SODs, has a protective role in the pathogenesis of superoxide radical–mediated brain injury, including cold injury–induced brain edema, traumatic brain injury, and focal transient ischemia/reperfusion.7 8 9 10
The global ischemia model is well known as a model corresponding to clinical situations such as cardiac arrest and external circulation in cardiovascular surgery. The hippocampus, particularly the CA1 subregion, is known to be one of the brain areas vulnerable to transient cerebral global ischemia. The postischemic interval required for the apparent selective neuronal loss is inversely proportional to the severity or duration of the ischemic insult, the so-called maturation phenomenon described by Ito et al.11 This interval is especially prolonged in the hippocampal neurons,12 a process called delayed neuronal death,13 although its detailed mechanism is still unclear. An apoptotic pathway was reported to be involved in the mechanism of hippocampal injury after transient global ischemia.14 15 The mechanism underlying apoptosis is multifactorial and includes reactive oxygen species as a major mediator.16 17 18 Our recent studies showed the increased production of O2- after global ischemia in the vulnerable hippocampal CA1 subregion.19 The reduction of hippocampal injury after transient global ischemia in transgenic mice and rats further implicates the determinate role of overexpressed human CuZn-SOD in global ischemic injury.20 21
The development of mice deficient in the mouse CuZn-SOD gene (Sod1) has provided a model for assessing the role of endogenous CuZn-SOD in nervous system injury.22 These mutant mice with a decrease in or a complete absence of endogenous CuZn-SOD activity were used in a dose-dependent study of CuZn-SOD activity in hippocampal injury after transient global ischemia. The present study was designed to clarify the protective role CuZn-SOD plays in the pathogenesis of selective vulnerability and delayed neuronal death after transient global ischemia.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
|---|
Sod1-Deficient Mice
Mutant mice designated as 129/CD1 –Sod1<tm1 Cep> were produced by Reaume et al22 at Cephalon, Inc (West Chester, Pa). These mutant mice were used to create mice that were either heterozygous (Sod1 +/-) or homozygous (Sod1 -/-) mutants. These mutant mice with the 129/CD1 background were bred with the CD1 strain of mice for at least 8 generations. There were no distinct phenotypes between these mutants and the wild-type animals. CuZn-SOD activity in the mutant mice was assessed by the method of nondenaturing gel electrophoresis assay, as previously described.23 These mutant mice and age-matched wild-type littermates generated from heterozygous parents (Sod1 +/-) were subjected to global ischemia as detailed below. All animals were treated in accordance with Stanford University guidelines and the animal protocol approved by Stanford University's Administrative Panel on Laboratory Animal Care.
Global Cerebral Ischemia
Global ischemia was induced by bilateral common carotid
artery (CCA) occlusion under controlled ventilation. Mice were
anesthetized with chloral hydrate (350 mg/kg IP) and xylazine
(4 mg/kg IP). The endotracheal tube was inserted, and respiration was
controlled with the use of an animal ventilator with inspiratory stroke
volume of 0.5 mL and a respiratory rate of 120 breaths per minute.
Rectal temperature was maintained at 37.0±0.5°C with a homeothermic
blanket. The bilateral CCA was exposed, and temporary clips were
applied. After a period of ischemia, the clips were removed,
and atropine sulfate (0.5 mg/kg) was injected
intraperitoneally to reverse respiratory and blood
pressure that were depressed by the chloral hydrate. The animals were
cared for in individual cages at 20°C.
Measuring Regional Cerebral Blood Flow
Change in regional cerebral blood flow (rCBF) was evaluated in
both groups of animals with a laser Doppler flowmeter (LASERFLO
BPM2, Vasomedic). A probe was placed on
the skull above the middle cerebral artery territory cortex (0.5
mm posterior and 4 mm lateral from bregma). The rCBF was monitored
continuously from 5 minutes before until 5 minutes after induction of
ischemia. Decreased rates of rCBF during bilateral CCA
occlusion were calculated as (ischemia
rCBF/preischemia rCBF)x100.
Carbon Black Evaluation of Plasticity of the Posterior
Communicating Arteries
Since the plasticity of the posterior communicating arteries
(PcomAs) would influence the outcome of hippocampal injury after
transient global ischemia, we assessed the plasticity of the
PcomAs before the histological evaluation of the
hippocampal injury. The animals were anesthetized with
ketamine and xylazine. After perfusion fixation through the
ascending aorta with 10 U/mL heparin in 0.9% saline and 3.7%
formaldehyde in PBS, carbon black ink in an equal volume of 20%
gelatin in H2O was injected. The removed brains
were fixed in 3.7% formaldehyde overnight at 4°C. Plasticity of
PcomAs was graded by a scale of 0 to 3. Since the contralateral PcomA
would make a collateral flow in the ipsilateral hippocampus, the
plasticity scores of the PcomAs in both hemispheres were totaled, then
the brains with scores of 0 to 3 were classified as the hypoplastic
PcomA group and the brains with sums >3 were classified as the normal
PcomA group.
In Situ Detection of Superoxide Production
The spatial production of superoxide anion
(O2-) in cerebral
ischemia was investigated by the method of in situ detection of
oxidized hydroethidium (Molecular Probes).24 25 26
Hydroethidium solution (200 µL; 1 mg/1 mL PBS with 1%
dimethylsulfoxide) was administered intravenously 1 hour
before the animals were killed. Hydroethidium rapidly penetrated into
the brain, which was selectively oxidized to ethidium by
O2-. Animals were killed 1 day
after global ischemia by transcardiac perfusion as
described above. After postfixation in 3.7% formaldehyde, brains were
sectioned to a 50-µm thickness at the level of the hippocampus with
the use of a vibratome and placed on glass slides (Superfrost, Fisher
Scientific). Fluorescence of ethidium was observed with a
microscope (Axioplan2, Zeiss) at excitation of 510 to 550 nm and
emission >580 nm. To analyze the fluorescence signal
of hydroethidium, photomicrographs (x630) were scanned by a GS-700
imaging densitometer (Bio-Rad), and then the signal intensity was
measured in 7 to 9 individual cells in each group with the use of
Multi-Analyst software (Bio-Rad).26
Histological Analysis of Hippocampal
Injury
Brain samples of the PcomA hypoplastic group were chosen for
histological analysis of hippocampal injury on
the basis that complete ischemia was induced without a supply
of collateral flow from posterior circulation. The brains were
sectioned to a 50-µm thickness with a vibratome and stained with
cresyl violet. Neuronal damage in the hippocampus was qualitatively
evaluated on the basis of a scoring system of 0
to 4, as described by Murakami et al21 : grade 0, no damage
to any hippocampal subregion; grade 1, scattered ischemic
neurons in CA1 subregion; grade 2, moderate ischemic damage in
CA1 subregion; grade 3, whole pyramidal cells damaged in
CA1 subregion; and grade 4, extensive cell damage in all hippocampal
subregions. Neural damage was evaluated by a researcher blinded to the
studies.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
|---|
CuZn-SOD Activity in Mutant Mice
Nondenatured gel electrophoresis followed by nitroblue tetrazolium staining confirms that CuZn-SOD activity is reduced in Sod1 +/- and completely absent in Sod1 -/- mutants (Figure 1
). These results were well matched with
our previous study in which we reported that the CuZn-SOD activity of
blood and brain tissue in this stain was reduced to 50% in
Sod1 +/- mutants and to only a trace in Sod1
-/- mutants.27
|
Physiological Conditions and Mortality
After Ischemia
The results of measuring rCBF showed a significant difference in
the decreased rate of rCBF during global ischemia between the
PcomA hypoplastic group (mean±SD, 6.8±5.5%) and the normoplastic
group (42.8±23.1%; n=10; P<0.01, ANOVA; Figure 2). However, within the hypoplastic
group, there was no significant difference in rCBF during
ischemia between wild types and Sod1 knockout
mutants. No significant difference was seen in PcomA plasticity between
the hypoplastic group of either genotype (Table 1). The mortality of each group is shown
in Table 2. No significant difference was
observed in the mortality data. Three days after 5 minutes of global
ischemia, the mortality was higher in the Sod1 +/-
mutants than in the wild types and was highest in the Sod1
-/- mutants. We did not perform 10 minutes of global ischemia
insult in the Sod1 homozygous knockout mutant mice because
we expected the mortality to be too high for these animals compared
with the 50% mortality with 5 minutes of global ischemia.
|
|
|
Production of O2- in the
Hippocampal CA1 Subregion
The expression of oxidized hydroethidium signals as a red color,
which was consistent with our previous report,25
shows production of O2-
in the hippocampal CA1 pyramidal cells after global
ischemia. Under normal physiological
conditions, oxidized hydroethidium signals were detected as small
particles in the cytosol, indicating the leakage of
O2- produced in the
mitochondria. One day after 5 minutes of global ischemia,
scattered cells in the Sod1 +/- mutants showed cytosolic
oxidized hydroethidium signals (Figure 3B); however, no marked changes were
observed in the wild types (Figure 3A). After more severe
ischemia, such as 10 minutes of global ischemia, the
red particles of oxidized hydroethidium signals were increased
slightly, but not the cytosolic expression, in the wild types (Figure 3C). On the other hand, almost all pyramidal cells
markedly showed cytosolic oxidized hydroethidium signals in the CA1
subregion of Sod1 +/- mice (Figure 3D). Mean
intensity of hydroethidium signal was significantly higher in mutant
mice 1 day after 5 minutes of ischemia (1.384±1.539; mean
optical density±SD) than in wild-type mice (0.194±0.143)
(P<0.05). After 10 minutes of ischemia,
hydroethidium signals in the CA1 subregion were significantly increased
in both groups, and mutant mice showed significantly higher signals
(4.619±0.704) than wild-type mice (1.902±1.393)
(P<0.001). The results that showed increased superoxide
radical signals in mutant mice in the postischemic CA1
subregion are considered to have occurred because of the 50% reduction
of Sod1 activity in the mutant mice compared with the
wild-type mice.27
|
Hippocampal Injury After Mild and Severe Global Ischemia
Various levels of severity of neuronal damage in the hippocampus
were seen after global ischemia, but the ischemic
damage was hardly observed in the brains of the group with normal
plasticity of the PcomA. To equalize the anatomic background between
Sod1 knockout mutants and wild types, the brains of the
PcomA hypoplastic group were used for histological
assessment of hippocampal injury after global ischemia.
Representative photographs showed morphological
features of injured brains and the hippocampus by cresyl violet
staining 3 days after 5 minutes of global ischemia in wild
types and Sod1 knockout mutants. Although
no remarkable change was observed in the wild types, ischemic
changes were observed in scattered neurons of the CA1 subregion in the
Sod1 +/- mice. Furthermore, severe damage and edema were
observed in the hippocampal CA1 subregion, dentate gyrus, and cortex in
Sod1 -/- mice (Figure 4). A
qualitative analysis shows neuronal damage of the hippocampus
in wild types and Sod1 knockout mutants 1 and 3 days after 5
and 10 minutes of global ischemia (Figure 5). No significant difference was
observed 1 day after 5 minutes of ischemia between each
genotype. Hippocampal damage was exacerbated in Sod1
+/- mutants and was significantly more severe in Sod1 -/-
mutants compared with wild types 3 days after 5 minutes of
ischemia. After 10 minutes of global ischemia,
hippocampal damage was greater in Sod1 +/- mutants than in
wild types at 1 day; however, there was no significant difference
between mutants and wild types 3 days after ischemia.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
|---|
Targeted disruption of the Sod1 gene results in the partial or complete loss of CuZn-SOD activity in mutant mice. This study shows that the level of production of O2- increased after global ischemia in the vulnerable hippocampal CA1 subregion, especially in the mutant mice with a reduction in CuZn-SOD activity. In addition, the present study demonstrated that the hippocampal injury was exacerbated in mutant mice after transient global ischemia. Furthermore, this protective effect of CuZn-SOD was obtained in both acute injury after relatively intense ischemia and in progressive injury after mild ischemia. These findings are consistent with the hypothesis that an increased level of O2- mediates these pathologies after transient global ischemia.
Gerbils have been widely used for global ischemia studies on
the basis that bilateral CCA occlusion can induce almost complete
forebrain ischemia without the reduction of collateral blood
flow from posterior circulation because of the lack of PcomAs as a
connection between anterior and posterior circulation. Several models
of global ischemia in mice have been proposed, such as 3-vessel
occlusion and unilateral occlusion with systemic hypoxia. We
previously reported on a simple mouse model of transient global
cerebral ischemia that is based on PcomA
distribution.21 28 In this report, we used this method
with 2 minor modifications. First, atropine sulfate was injected
intraperitoneally just after reperfusion to recover
blood pressure and respiration, which were suppressed by the chloral
hydrate. rCBF measured by laser Doppler flowmeter was not increased
to the basal level after release of bilateral CCA occlusion without the
atropine sulfate injection. Second, the previous method selectively
used the hippocampus with the hypoplastic PcomA in the ipsilateral
hemisphere to evaluate ischemic damage. However, even if the
PcomA was hypoplastic in the ipsilateral hemisphere, rCBF did not
decrease enough to make an ischemic change if the contralateral
PcomA was plastic as well. The blood flow would come through
azygos anterior cerebral arteries. Therefore, we totaled each
score of the bilateral PcomAs and classified them as PcomA hypoplastic
brains and normoplastic brains with the cutoff point at 3. We then used
the PcomA hypoplastic group to evaluate the hippocampal damage after
ischemia. The validity of this classification was confirmed by
measuring rCBF during ischemia. Using this method, we observed
a hippocampal injury after transient global ischemia in mutant
mice with a CuZn-SOD deficiency. The mean scores of PcomA plasticity
and the decreased levels of rCBF did not show a significant difference
between wild types and Sod1 knockout mutants (Table 1
), indicating that the ischemic condition induced by
the present method was at the same level in each group of
animals.
Our previous study showed the production of
O2- in the hippocampal CA1
subregion after global ischemia in rats with the use of in situ
imaging of superoxide by hydroethidium oxidation.20 It was
then suspected that production of
O2- increased in mutant mice
with CuZn-SOD deficiency. In the present study, we clearly show
that the O2- signals increased
in Sod1 mutants compared with wild types. As previously
observed,25 29
O2- signals were shown by
oxidized hydroethidium as small particles in the cytosol, suggesting
mitochondrial production of
O2-. Under normal conditions,
these signals were observed without any significant difference in
either group (data not shown). After mild ischemia, diffuse
cytosolic expression of O2-
signals was observed in scattered cells in the hippocampal
pyramidal cells in Sod1 +/- mice (Figure 2B); therefore, after severe ischemia, a marked, diffuse
cytosolic expression of O2-
signals was seen in pyramidal cells in the CA1 subregion of
Sod1 +/- mutants (Figure 3D). On the other hand, a
particle-like expression of O2-
signals increased, but a diffuse expression was not observed in wild
types (Figure 3C). These findings indicate that
O2-, which was overproduced in
the mitochondria and leaked into the cytosol, was trapped by CuZn-SOD
in wild types. However, in Sod1 mutant mice, it exceeded the
capacity of CuZn-SOD, and then the
O2- signals filled the cytosol
of pyramidal cells in the vulnerable hippocampal CA1
subregion after global ischemia.
Mutant mice with a CuZn-SOD deficiency exhibited a significant increase
in damage in a dose-dependent manner in the hippocampus after transient
global cerebral ischemia. Hippocampal damage tended to be
exacerbated in Sod1 mutants but was not significant 1 day
after relatively mild ischemia (5 minutes). However, the
mortality (Table 2) and the hippocampal damage (Figures 4 and 5) were markedly exacerbated in reverse proportion to
CuZn-SOD activity 3 days after mild ischemia. With these data,
we propose that CuZn-SOD plays a protective role in delayed hippocampal
damage after mild global ischemia, which conforms to the
results of global ischemia in CuZn-SOD transgenic
rats.20 However, after 10 minutes of global
ischemia, which is a relatively severe ischemia, the
hippocampal damage was significantly exacerbated in Sod1
+/- mutants 1 day after ischemia, although severe damage was
observed in wild types 3 days after ischemia. These findings
suggest that CuZn-SOD has a protective effect against acute damage
after severe ischemic insult, and these findings are supported
by the results we previously reported showing that neuronal damage was
decreased in Sod1 transgenic mice after focal
ischemia8 10 and was exacerbated in
Sod1 knockout mutants.27 Since
Sod1 was reported to delay neuronal apoptosis in
vitro,30 we do not completely rule out the
possibility that the earlier development of the hippocampal damage in
Sod1 knockouts may contribute, in part, to the marked
exacerbation of the hippocampal injury after global ischemia in
the present study (Figure 5). A more detailed time course
study would address this important issue.
In the present study, the mechanisms of O2- that mediate hippocampal injury after transient global ischemia are still unclear. Recent studies, although still somewhat controversial, have identified some apoptotic features by biochemical and morphological evidence such as terminal deoxynucleotidyl transferase–mediated dUTP biotin nick end labeling (TUNEL) staining14 31 32 and internucleosomal DNA fragmentation, as indicated by the DNA laddering pattern.33 Several apoptosis-regulatory genes and enzymes are found to be induced in ischemic cells. Caspase-3, which is in the interleukin 1ß converting enzyme family of cysteine proteases, was reported to have an important effect in apoptotic cell death. The caspase-3 mRNA and activated form of the protein were predominantly increased in hippocampal CA1 neurons after transient global ischemia. Furthermore, ventricular infusion of z-DEVD-fmk, a caspase-3 inhibitor, decreased cell death and DNA fragmentation in the CA1 subregion after global ischemia.34
We have previously reported that Sod1 knockouts showed a marked increase of the amount of DNA fragmentation and of infarction volume after transient focal cerebral ischemia in mice27 and that DNA damage was reduced in the hippocampal CA1 subregion in Sod1 transgenic rats after global ischemia,20 together suggesting that reactive oxygen species could exacerbate DNA damage and infarction after ischemia/reperfusion. In the present study, we have shown the first evidence of the dose-dependent effect of endogenous SOD on the increase of hydroethidium signals and on the hippocampal injury after global cerebral ischemia. As for DNA damage after global ischemia, our recent study showed that the downregulation of the DNA repair enzyme apurinic/apyrimidinic endonuclease (APE/Ref-1), which plays a central role in the base excision repair pathway by providing a 3'-OH primer for repair synthesis of DNA after all types of oxidative damage,35 is implicated in apoptotic cell death after global ischemia.19 Although the cell death pathway, through either necrosis or apoptosis, is still unclear after transient global ischemia, DNA fragmentation was observed after global ischemia insults. These data again suggest that oxidative DNA damage may cause hippocampal damage after global ischemia. Future studies, such as DNA damage indicated by TUNEL staining, DNA laddering, caspase-3 activation, and downregulation of APE/Ref-1 expression in Sod1 knockout mice after transient global ischemia, are considered necessary to address this critical issue.
In conclusion, selective knockout of Sod1 gene reduced CuZn-SOD activity and decreased neuronal damage exacerbated by CuZn-SOD activity in the hippocampal CA1 subregion after transient global ischemia. Our results support the hypothesis that CuZn-SOD plays an important role in neuronal damage in vulnerable regions after transient global ischemia. Overexpression of CuZn-SOD by gene transfer may have therapeutic implications in preventing delayed neuronal damage in patients who sustain stroke or cardiac arrest.
|
Acknowledgments |
|---|
This work was supported by National Institutes of Health grants NS14543, NS25372, NS36147, NS38653, and NO1 NS82386. Dr Chan is a recipient of the Jacob Javits Neuroscience Investigator Award. We thank L. F. Reola, B.E. Calagui, and S.F. Chen for their expert technical assistance and C. Christensen for editorial assistance.
Received March 2, 1999; revision received May 6, 1999; accepted June 7, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
|---|
1. Kontos HA. George E. Brown memorial lecture: oxygen radicals in cerebral vascular injury. Circ Res. 1985;57:508–516.
2.
Coyle JT, Puttfarcken P. Oxidative stress, glutamate,
and neurodegenerative disorders. Science. 1993;262:689–695.
3. Chan PH. Oxygen radicals in focal cerebral ischemia. Brain Pathol. 1994;4:59–65.[Medline] [Order article via Infotrieve]
4.
Chan PH. Role of oxidants in ischemic brain
damage. Stroke. 1996;27:1124–1129.
5. Fridovich I. Biological effects of the superoxide radical. Arch Biochem Biophys. 1986;247:1–11.[Medline] [Order article via Infotrieve]
6. Patel M, Day BJ, Crapo JD, Fridovich I, McNamara JO. Requirement for superoxide in excitotoxic cell death. Neuron. 1996;16:345–355.[Medline] [Order article via Infotrieve]
7. Chan PH, Yang GY, Chen SF, Carlson E, Epstein CJ. Cold-induced brain edema and infarction are reduced in transgenic mice overexpressing CuZn-superoxide dismutase. Ann Neurol.. 1991;29:482–486.[Medline] [Order article via Infotrieve]
8.
Kinouchi H, Epstein CJ, Mizui T, Carlson E, Chen 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.
9. Mikawa S, Kinouchi H, Kamii H, Gobbel GT, Chen SF, Carlson E, Epstein CJ, Chan PH. Attenuation of acute and chronic damage following traumatic brain injury in copper, zinc-superoxide dismutase transgenic mice. J Neurosurg. 1996;85:885–891.[Medline] [Order article via Infotrieve]
10. Yang G, Chan PH, Chen J, Carlson E, Chen SF, Weinstein P, Epstein CJ, Kamii H. Human copper-zinc superoxide dismutase transgenic mice are highly resistant to reperfusion injury after focal cerebral ischemia. Stroke. 1994;25:165–170.[Abstract]
11. Ito U, Spatz M, Walker JJ, Klatzo I. Experimental cerebral ischemia in Mongolian gerbils, I: light microscopic observations. Acta Neuropathol (Berl). 1975;32:209–223.[Medline] [Order article via Infotrieve]
12. Pulsinelli WA, Brierley JB, Plum F. Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann Neurol. 1982;11:491–498.[Medline] [Order article via Infotrieve]
13. Kirino T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 1982;239:57–69.[Medline] [Order article via Infotrieve]
14. MacManus JP, Buchan AM, Hill IE, Rasquinha I, Preston E. Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain. Neurosci Lett. 1993;164:89–92.[Medline] [Order article via Infotrieve]
15. Nitatori T, Sato N, Waguri S, Karasawa Y, Araki H, Shibanai K, Kominami E, Uchiyama Y. Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis. J Neurosci. 1995;15:1001–1011.[Abstract]
16. Greenlund L. Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death. Neuron. 1995;14:303–315.[Medline] [Order article via Infotrieve]
17. Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell. 1993;75:241–245.[Medline] [Order article via Infotrieve]
18.
Kane DJ, Sarafian TA, Anton R, Hahn H, Gralla EB,
Valentine JS, Ord T, Bredesen DE. Bcl-2 inhibition of neural death:
decreased generation of reactive oxygen species. Science. 1993;262:1274–1277.
19.
Kawase M, Fujimura M, Morita-Fujimura Y, Chan PH.
Reduction of apurinic/apyrimidinic endonuclease expression after
transient global cerebral ischemia in rats: implication of the
failure of DNA repair in neuronal apoptosis. Stroke. 1999;30:441–449.
20.
Chan PH, Kawase M, Murakami K, Chen SF, Li Y, Calagui
B, Reola L, Carlson E, Epstein CJ. Overexpression of SOD1 rats protects
vulnerable neurons against ischemic damage after global
cerebral ischemia and reperfusion. J Neurosci. 1998;18:8292–8299.
21.
Murakami K, Kondo T, Epstein CJ, Chan PH.
Overexpression of CuZn-superoxide dismutase reduces hippocampal injury
after global ischemia in transgenic mice. Stroke. 1997;28:1797–1804.
22. Reaume AG, Elliott JL, Hoffman EK, Kowall NW, Ferrante RJ, Siwek DF, Wilcox HM, Flood DG, Beal MF, Brown RJ, Scott RW, Snider WD. Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury. Nat Genet. 1996;13:43–47.[Medline] [Order article via Infotrieve]
23.
Epstein CJ, Avraham KB, Lovett M, Smith S, Elroy SO,
Rotman G, Bry C, Groner Y. Transgenic mice with increased
Cu/Zn-superoxide dismutase activity: animal model of dosage effects in
Down syndrome. Proc Natl Acad Sci U S A. 1987;84:8044–8048.
24.
Bindokas VP, Jordan J, Lee CC, Miller RJ. Superoxide
production in rat hippocampal neurons: selective imaging with
hydroethidine. J Neurosci. 1996;16:1324–1336.
25.
Murakami K, Kondo T, Kawase M, Li Y, Sato S, Chen SF,
Chan PH. Mitochondrial susceptibility to oxidative stress exacerbates
cerebral infarction that follows permanent focal cerebral
ischemia in mutant mice with manganese superoxide dismutase
deficiency. J Neurosci. 1998;18:205–213.
26.
Fujimura M, Morita-Fujimura Y, Kawase M, Copin J-C,
Calagui B, Epstein CJ, Chan PH. Manganese superoxide dismutase mediates
the early release of mitochondrial cytochrome c and subsequent DNA
fragmentation after permanent focal cerebral ischemia in mice.
J Neurosci.. 1999;19:3414–3422.
27.
Kondo T, Reaume AG, Huang TT, Carlson E, Murakami K,
Chen SF, Hoffman EK, Scott RW, Epstein CJ, Chan PH. Reduction of
CuZn-superoxide dismutase activity exacerbates neuronal cell injury and
edema formation after transient focal cerebral ischemia.
J Neurosci. 1997;17:4180–4189.
28. Murakami K, Kondo T, Kawase M, Chan PH. The development of a new mouse model of global ischemia: focus on the relationships between ischemia duration, anesthesia, cerebral vasculature, and neuronal injury following global ischemia in mice. Brain Res. 1998;780:304–310.[Medline] [Order article via Infotrieve]
29. Kondo T, Li Y, Sato S, Murakami K, Copin J, Huang T, Epstein C, Chan P. Subcellular localization of superoxide anions following focal cerebral ischemia and reperfusion. J Cereb Blood Flow Metab. 1997;17(suppl 1):S102. Abstract.
30. Greenlund LJ, Deckwerth TL, Johnson EM Jr. Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death. Neuron.. 1995;14:303–315.
31. Sei Y, Von Lubitz KJ, Basile AS, Borner MM, Lin RC, Skolnick P, Fossom LH. Internucleosomal DNA fragmentation in gerbil hippocampus following forebrain ischemia. Neurosci Lett. 1994;171:179–182.[Medline] [Order article via Infotrieve]
32. Petito CK, Torres-Munoz J, Roberts B, Olarte J-P, Nowak TS Jr, Pulsinelli WA. DNA fragmentation follows delayed neuronal death in CA1 neurons exposed to transient global ischemia in the rat. J Cereb Blood Flow Metab. 1997;17:967–976.[Medline] [Order article via Infotrieve]
33. Heron A, Pollard H, Dessi F, Moreau J, Lasbennes F, Ben-Ari Y. Regional variability in DNA fragmentation after global ischemia evidenced by combined histological and gel electrophoresis observation in the rat brain. J Neurochem. 1993;61:1973–1976.[Medline] [Order article via Infotrieve]
34.
Chen J, Nagayama T, Jin K, Stetler RA, Zhu RL, Graham
SH, Simon RP. Induction of caspase-3-like protease may mediate delayed
neuronal death in the hippocampus after transient cerebral
ischemia. J Neurosci. 1998;18:4914–4928.
35. Mitra S, Hazra TK, Roy R, Ikeda S, Biswas T, Lock J, Boldogh I, Izumi T. Complexities of DNA base excision repair in mammalian cells. Mol Cells. 1997;7:305–312.[Medline] [Order article via Infotrieve]
Editorial Comment
Department of Physiology, New York Medical College, Valhalla, New York
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
|---|
The accompanying article from Kawase and collaborators uses mutant mice deficient in the cytosolic or CuZn form of SOD to demonstrate the important dose-dependent role of this enzyme in protecting the hippocampal CA1 subregion from the delayed injury caused by exposure to a transient (5- to 10-minute) global ischemia produced by occlusion of the CCA. While there is already much evidence supporting the importance of superoxide in the injury caused by exposure of the hippocampus to ischemia and reperfusion and the protective role of CuZn-SOD, the use of homozygous (Sod1 -/-) and heterozygous (Sod1 +/-) mice with a complete absence and a 50% decrease in CuZn-SOD activity, respectively, permitted the demonstration of the concentration-dependent effects of this enzyme in protection against hippocampal damage.
There appear to be at least several key processes through which superoxide anion can participate in tissue injury. First, superoxide anion is a key source of production of more reactive oxidants that directly cause damage to key organelles and constituents of tissues. Highly reactive oxidant species can result from processes including the generation of peroxynitrite by the reaction of superoxide with nitric oxide and from the reductive release of bound iron by superoxide and the subsequent generation of hydroxyl radical–like oxidants from reactions of ferrous iron with peroxides. In addition, superoxide anion appears to cause mitochondrial dysfunction associated with the disruption of iron-sulfur centers in the electron transport chain. This and other actions of superoxide-derived oxidants appear to be key processes that result in apoptosis. Thus, superoxide anion has multiple ways of promoting tissue injury processes.
CuZn-SOD is one of the 3 major types of SOD that are present in mammalian tissues. This form of SOD is primarily located in the cytosol of cells, and its principal function appears to be the removal of superoxide from this intracellular compartment. The Mn form of SOD is essentially a mitochondrial enzyme, and it is thought to metabolize superoxide generated within the mitochondria in a manner that efficiently prevents the detection of superoxide release from this organelle. The third form of SOD is an extracellular enzyme (EC-SOD), which is thought to scavenge superoxide in the extracellular environment of cells that secrete this enzyme. While there is evidence that superoxide can be transported across membranes, the extent to which the scavenging of superoxide made in one compartment by SOD that is present in another compartment provides protection against aspects of cellular injury is an area that is very poorly understood. There is substantial evidence that each form of SOD participates in the protection of tissues from the pathophysiological effects of oxidant stress in the regions where these enzymes are located. This suggests that each form of SOD is likely to have protective roles against aspects of tissue injury processes activated by ischemia and reperfusion. The results of the present study demonstrate the important protective role of the scavenging of superoxide by the CuZn form of SOD, which is located primarily in the cytosol of hippocampal cells.
Received March 2, 1999; revision received May 6, 1999; accepted June 7, 1999.
This article has been cited by other articles:
 |
|
 |
|
  D. P. D'Agostino, R. W. Putnam, and J. B. Dean Superoxide ({middle dot}O2 ) Production in CA1 Neurons of Rat Hippocampal Slices Exposed to Graded Levels of Oxygen J Neurophysiol, August 1, 2007; 98(2): 1030 - 1041. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-H. Zhu, X. Zhang, J. P. McClung, and X. G. Lei Impact of Cu,Zn-Superoxide Dismutase and Se-Dependent Glutathione Peroxidase-1 Knockouts on Acetaminophen-Induced Cell Death and Related Signaling in Murine Liver Experimental Biology and Medicine, December 1, 2006; 231(11): 1726 - 1732. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. Park, W.-N. Qi, Y. Cai, I. Zelko, J. Q. Liu, L.-E. Chen, J. R. Urbaniak, and R. J. Folz Skeletal muscle reperfusion injury is enhanced in extracellular superoxide dismutase knockout mouse Am J Physiol Heart Circ Physiol, July 1, 2005; 289(1): H181 - H187. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Tummala, C. Jung, A. Tiwari, C. M. J. Higgins, L. J. Hayward, and Z. Xu Inhibition of Chaperone Activity Is a Shared Property of Several Cu,Zn-Superoxide Dismutase Mutants That Cause Amyotrophic Lateral Sclerosis J. Biol. Chem., May 6, 2005; 280(18): 17725 - 17731. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zhao, J. Joseph, H. M. Fales, E. A. Sokoloski, R. L. Levine, J. Vasquez-Vivar, and B. Kalyanaraman Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence PNAS, April 19, 2005; 102(16): 5727 - 5732. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-R. Lee, K. Tsuji, S.-R. Lee, and E. H. Lo Role of Matrix Metalloproteinases in Delayed Neuronal Damage after Transient Global Cerebral Ischemia J. Neurosci., January 21, 2004; 24(3): 671 - 678. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. P. Didion, M. J. Ryan, L. A. Didion, P. E. Fegan, C. D. Sigmund, and F. M. Faraci Increased Superoxide and Vascular Dysfunction in CuZnSOD-Deficient Mice Circ. Res., November 15, 2002; 91(10): 938 - 944. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||