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(Stroke. 1996;27:1124-1129.)
© 1996 American Heart Association, Inc.

Articles

Role of Oxidants in Ischemic Brain Damage

Pak H. Chan, PhD

From the Departments of Neurological Surgery and Neurology, University of California, School of Medicine, San Francisco.

Correspondence to Pak H. Chan, PhD, CNS Injury and Edema Research Center, University of California, 521 Parnassus, C-224, San Francisco, CA 94143-0651.

	Abstract

Top Abstract Introduction Oxygen Radical Production in... Antioxidant Enzyme SODs and... Transgenic and Knockout Mutant... Conclusion References

 
Background and Purpose Oxygen free radicals or oxidants have been proposed to be involved in acute central nervous system injury that is produced by cerebral ischemia and reperfusion. Because of the transient nature of oxygen radicals and the technical difficulties inherent in accurately measuring their levels in the brain, experimental strategies have been focused on the use of pharmacological agents and antioxidants to seek a correlation between the exogenously supplied specific radical scavengers (ie, superoxide dismutase and catalase) and the subsequent protection of cerebral tissues from ischemic injury. However, this strategy entails problems (hemodynamic, pharmacokinetic, toxicity, blood-brain barrier permeability, etc) that may cloud the data interpretation. This mini-review will focus on the oxidant mechanisms in cerebral ischemic brain injury by using transgenic and knockout mice as an alternative approach.

Methods Transgenic and knockout mutants that either overexpress or are deficient in antioxidant enzyme/protein levels have been successfully produced. The availability of these genetically modified animals has made it possible to investigate the role of certain oxidants in ischemic brain cell damage in molecular fashion.

Results It has been shown that an increased level of CuZn–superoxide dismutase and antiapoptotic protein Bcl-2 in the brains of transgenic mice protects neurons from ischemic/reperfusion injury, whereas a deficiency in CuZn–superoxide dismutase or mitochondrial Mn–superoxide dismutase exacerbates ischemic brain damage. Target disruption of neuronal nitric oxide synthase in mice also provides neuronal protection against permanent and transient focal cerebral ischemia.

Conclusions I conclude that molecular genetic approaches in modifying antioxidant levels in the brain offer a unique tool for understanding the role of oxidants in ischemic brain damage.

Key Words: cerebral ischemia • free radicals • oxidants • reperfusion • superoxide dismutase • mice, transgenic • mice, knockout

	Introduction

Top Abstract Introduction Oxygen Radical Production in... Antioxidant Enzyme SODs and... Transgenic and Knockout Mutant... Conclusion References

 
Oxygen free radicals or oxidants have been implicated in the development of many neurological disorders and brain dysfunctions.^{1 2 3} One role of oxygen free radicals in brain injury appears to involve reperfusion after cerebral ischemia. In either global or focal cerebral ischemia, cerebral blood flow is significantly reduced in the brain regions that are supplied with oxygen by the occluded vessel. Reoxygenation during reperfusion provides oxygen to sustain neuronal viability and also provides oxygen as a substrate for numerous enzymatic oxidation reactions that produce reactive oxidants. In addition, reflow after occlusion often causes an increase in oxygen to levels that cannot be utilized by mitochondria under normal physiological flow conditions (Figure). It has been demonstrated that about 2% to 5% of the electron flow in isolated brain mitochondria produces superoxide radicals and H₂O₂.⁴ These constantly produced oxygen radicals are scavenged respectively by SODs and by GSHPx and catalase. Other antioxidants, including glutathione, ascorbic acid, and vitamin E, are also likely to be involved in the detoxification of free radicals. During reperfusion, perturbation of the antioxidative defense mechanisms is a result of the overproduction of oxygen radicals, inactivation of detoxification systems, consumption of antioxidants, and failure to adequately replenish antioxidants in the ischemic brain tissue. Because of technical difficulties, the level of oxygen free radicals in brain tissue after ischemia is generally assessed by indirect methods, including lipid peroxidation, protein oxidation, and DNA damage (Figure). Recent advances in methodologies have allowed investigators to measure hydroxyl radicals by salicylate hydroxylation⁵ and NO radicals with a porphyrinic microsensor⁶ and with electron paramagnetic resonance spin trapping⁷ in ischemic brain tissue. In addition, several chemical and biochemical methods have been developed to measure superoxide radicals in neurons that undergo oxidative stress.^{8 9 10 11} Despite these useful measurements for superoxide, hydroxyl, and NO radicals, establishment of the causative role of oxygen radicals in ischemic brain injury remains a difficult task for stroke researchers and neuroscientists. This review will attempt to assess the role of oxygen radicals in cerebral ischemia and the molecular genetic approaches using transgenic and knockout mutant mice and to dissect out the molecular and cellular mechanisms involving oxygen radicals in ischemic brain damage.

View larger version (9K): [in this window] [in a new window]   Figure 1. Oxygen radicals in cerebral ischemia and reperfusion injury. Mt indicates mitochondria; Fe2+, ferrous iron.

	Oxygen Radical Production in Ischemic Brain

Top Abstract Introduction Oxygen Radical Production in... Antioxidant Enzyme SODs and... Transgenic and Knockout Mutant... Conclusion References

 
Several oxygen radical species, including O₂·^-, HO₂·, H₂O₂, and ·OH, are formed following the initial reduction of oxygen:

(1)

(2)

(3)

(4)

In Equation 3, SOD catalyzes this reaction at physiological pH at an extremely fast, constant rate (2x10⁹ L·mol^-1), forming H₂O₂ that is then detoxified to H₂O and O₂ by catalase in mammalian cells or in the brain by GSHPx at the expense of reduced glutathione (Equation 4). The oxidized glutathione can be recycled to reduced glutathione by glutathione reductase in the presence of nicotinamide-adenine dinucleotide phosphate.

Hydroxyl radicals are extremely active oxidants. These radicals are known to initiate lipid peroxidation and to cause protein oxidation and DNA damage in cells (Figure). Superoxide radicals, on the other hand, are much less reactive but have a longer half-life and can form hydroxyl radicals through a Haber-Weiss reaction (see Equation 5).

(5)

(6)

(7)

(8)

It is generally believed that this reaction (Equation 5) proceeds rapidly in the presence of trace metal iron (Fenton reaction). Another pathway for forming ·OH is through the reaction of O₂·^- and NO· (a gas radical that is constantly being formed in the brain by neuronal, endothelial, and glial NOS).¹² The product of this reaction is ONOO^-.¹³

This reaction (Equation 6) is extremely rapid, and the constant rate of ONOO^- formation (6.7x10⁹ L·mol^-1·s^-1) is diffusion-limited.¹⁴ At physiological pH, ONOO^- degrades instantly to ·OH and nitrogen dioxide (NO₂·) (see Equation 8). ONOO^-, a stronger pro-oxidant, can react with SOD to form a powerful nitrating agent, resulting in the nitration of tyrosines of cellular proteins and initiation of cellular dysfunction and death (Figure).¹⁵ However, the role of ONOO^- in ischemic brain injury has only recently been investigated. On the other hand, contradictory reports have surfaced with regard to the role of NO· in ischemic neuronal injury, primarily because of its dual role as a vasodilator (ie, increases in cerebral blood flow)¹⁶ and as a free radical that can injure neuronal cells.¹⁷ Other studies have suggested that the redox state of NO· may determine its role as either neuronal protector or injurious mediator after activation of N-methyl-D-aspartate receptors.¹⁸ It is of interest to note that downregulation of CuZn-SOD activity in PC12 cells by exposure to antisense oligonucleotides leads to apoptotic cell death via the NO·-ONOO^- pathway.¹⁹

	Antioxidant Enzyme SODs and Cerebral Ischemia

Top Abstract Introduction Oxygen Radical Production in... Antioxidant Enzyme SODs and... Transgenic and Knockout Mutant... Conclusion References

 
Several enzymes, including SOD, GSHPx, glutathione reductase, and catalase, are endogenous antioxidants that process specific free radical scavenging properties. SODs, in particular, have been used extensively to reduce superoxide radical–associated ischemic brain damage.

Based on the metal ion requirements and the anatomic distribution, three types of SODs exist in brain cells. CuZn-SOD is a cytosolic enzyme that requires both copper and zinc ions as cofactors. It is a dimeric protein that is coded by the human CuZn-SOD transgene (SOD-1) on chromosome 21 in human cells. Manganese (Mn-SOD) is a mitochondrial enzyme with requirements for Mn²⁺.²⁰ It is a tetrameric protein that is coded by the SOD-2 gene in chromosome 4 in human cells. Both CuZn-SOD and Mn-SOD from various sources have been fully characterized biochemically, and the cDNAs of both human enzymes have been successfully cloned.²¹ A copper-containing SOD (sod-3) has been identified in the extracellular space, and its gene has been successfully cloned (Table 1).²² As specified for superoxide radicals, CuZn-SOD has been used extensively to reduce brain injury induced by ischemia and reperfusion. Unfortunately, investigators have obtained various degrees of success and failure when free nonmodified SOD was used to ameliorate ischemic brain injury.²³ The extremely short half-life of CuZn-SOD (6 minutes) in circulating blood and its failure to pass the blood-brain barrier make it difficult to use enzyme therapy in cerebral ischemia. However, a modified enzyme with an increased half-life, such as polyethylene glycol–conjugated SOD, has been successfully used to reduce infarct volume in rats that have been subjected to focal cerebral ischemia.²⁴ Liposome-entrapped SOD has an increased half-life (4.2 hours), blood-brain barrier permeability, and cellular uptake, and it has also proved to be an effective treatment in reducing the severity of traumatic and focal ischemic brain injuries.^{25 26} Yet, in some instances, modified SOD (ie, polyethylene glycol–conjugated SOD) has been used with conflicting results.²⁴ The fact that the results are mixed make it imperative to use other experimental strategies so the role of SOD can be fully established in cerebral ischemia.

View this table: [in this window] [in a new window]   Table 1. Mammalian Superoxide Dismutases

	Transgenic and Knockout Mutant Mice as Useful Tools to Study the Role of Oxidants in Ischemic Brain Injury

Top Abstract Introduction Oxygen Radical Production in... Antioxidant Enzyme SODs and... Transgenic and Knockout Mutant... Conclusion References

 
One strategy is to use transgenic/knockout technology to alter the levels of pro-oxidants, antioxidants, and oxidant-related enzymes or proteins and to use these transgenic animals to study the role of a specific oxidant or antioxidant in ischemic brain injury.

The most commonly used method for creating transgenic mice is the microinjection of DNA of interest (transgene) into the pronuclei of fertilized mouse oocytes.²⁷ This technique is used to introduce a linear DNA fragment (genomic or cDNA with proper promoter) that contains sequences required for directing correct transcriptional initiation and for encoding a gene product (protein, enzyme) that can be readily identified using various biochemical assays. Pronuclear microinjection of DNA leads to random integration of various copies of the injected DNA into a single site on a mouse chromosome. The integrated DNA can be passed on to future generations. Another way to produce transgenic mice is to introduce foreign genes to embryonic stem cells.²⁸ The embryonic stem cells are subsequently screened for the integration of transgenes. These embryonic stem cells with the transgene (active or inactive knockout) can then be microinjected into the blastocytes to further produce chimeric mice. The mating of chimeric mice with wild types produces heterozygous offspring (+/-) that contain the transgene. Furthermore, breeding among heterozygous mice generates homozygous animals (+/+ for overexpression, -/- for knockout). By use of this transgenic technology, several mice strains with various genotypes that relate to oxidant/antioxidant enzymes/proteins have been successfully developed. Most of these transgenic and knockout mutant animals have been used in the study of the role of oxidants in ischemic brain injury (Table 2).

View this table: [in this window] [in a new window]   Table 2. Transgenic and Knockout Mutant Mice in the Study of the Role of Oxidants in Ischemic Brain Injury

In the studies using transgenic mice overexpressing SOD-1,⁴² two models of focal cerebral ischemia are used. One model is achieved by permanent occlusion of the left MCA just proximal to the piriform branch of the anesthetized mouse. Immediately after occlusion of the MCA, the left CCA is permanently ligated, and the right CCA is temporarily occluded for 1 hour followed by additional reperfusion for 24 hours. The total infarct volume (in cubic millimeters) is reduced by 36% in SOD-1 transgenic mice compared with nontransgenic mice. The brain edema is also reduced in both the MCA territory and the anterior cerebral artery area, a region that corresponds to the "ischemic penumbra." Antioxidants, both reduced glutathione and ascorbic acid, are maintained at higher levels in the penumbra area than in the same anatomic regions in nontransgenic mouse brains.²⁹ In another study that involved the use of SOD-1 transgenic mice, MCA occlusion was achieved with a 5-0 rounded nylon suture placed within the internal cerebral artery followed by withdrawal of the suture to allow reperfusion. The infarct volume was reduced by 26% in SOD-1 transgenic mice at 3 hours of reperfusion after 3 hours of ischemia³⁰ and by 40% in SOD-1 transgenic mice compared with nontransgenic mice at 24 hours after 1 hour of MCA occlusion.⁴³ The decrease in the infarct volume in SOD-1 transgenic mice clearly parallels the reduction of neurological deficits and is not related to the changes in cerebral blood flow. On the other hand, the infarct volume produced in SOD-1 transgenic mice that are subjected to 24 hours of permanent MCA occlusion using intraluminal suture blockade is not different from that in nontransgenic mice,³¹ suggesting that the increased ischemic severity resulting from prolonged cerebral ischemia will limit the protective role of CuZn-SOD against neuronal injury.

In addition to the SOD-1 transgenic mice, transgenic mice overexpressing bFGF when subjected to right CCA ligation, hyperglycemia, and 20 minutes of 1% carbon monoxide contained more viable hippocampal CA1 neurons than nontransgenic mice.³³ Although the mechanism by which the bFGF protects hippocampal CA1 is unknown, it is possible that increased antioxidant enzyme activities in hippocampal neurons by bFGF overexpression⁴⁴ may account for the neuronal protection.⁴⁵ Transgenic mice in which neurons overexpress the human BCL-2 protein under the control of the neuron-specific enolase or phosphoglycerate kinase promoters are protected against cerebral ischemia produced by permanent MCA occlusion.³² BCL-2, a membranous protein that is encoded by bcl-2, a mammalian homologue of the nematode antiapoptotic gene ced-9,⁴⁶ has been known to interfere with neuronal apoptosis by acting as an antioxidant.^{47 48 49} It has been demonstrated that the neurons that survive focal cerebral ischemic insult also express BCL-2 protein.⁵⁰

In a recent study, Lawrence et al⁵¹ demonstrated that overexpression of BCL-2 with herpes simplex virus vectors protects the central nervous system neurons against focal cerebral ischemia in rats and against glutamate-induced injury in hippocampal neurons in culture. Thus, these studies enforce the antioxidative concept in cerebral ischemia, since both transgenic mice and rats overexpressing antioxidant protein BCL-2 are more resistant to cerebral ischemia–induced brain damage.

On the other hand, a limited number of mutant mice that contain a null mutation of a specific gene have been developed and used for the study of molecular and cellular mechanisms of cerebrovascular diseases. It has been demonstrated that ischemic infarction is significantly reduced in neuronal NOS knockout mice³⁵ that were developed by Huang et al,⁵² clearly indicating that NO radicals produced by neuronal NOS are major determinants of the pathogenesis of the infarct after permanent focal ischemia. These same investigators have recently developed knockout mice that are deficient in endothelial NOS.^{36 37} Inducible NOS has been implicated in cerebral ischemic damage,⁵³ and mutant mice deficient in inducible NOS have been successfully produced.³⁸ It is anticipated that the role of inducible NOS in cerebral ischemia can be fully elucidated by using these mutant mice. In another study using knockout mice, the ischemic infarct volume was significantly reduced in p53 mutant mice (both homozygous and heterozygous) after focal cerebral ischemia compared with the wild type.³⁹ Since the loss of p53 tumor suppressor gene has been shown to protect neurons from kainate-induced cell death,⁵⁴ the neuronal protection offered by p53-deficient mutants may be related to the reduction of oxidative stress induced by kainate.⁵⁵

Using the knockout mutant mice, preliminary studies have been carried out to address the role of mouse cytosolic CuZn-SOD (sod-1) and Mn-SOD (sod-2)⁴⁰ in ischemic brain injury. It has recently been shown that ischemic infarct is significantly increased in mice that lack the sod-1 (homozygous and heterozygous) or sod-2 (heterozygous) gene.⁴¹ These studies, albeit preliminary, did point to the involvement of superoxide radicals in ischemic brain damage and to the important role these antioxidant enzymes play in neuronal protection after a focal ischemic insult.

	Conclusion

Top Abstract Introduction Oxygen Radical Production in... Antioxidant Enzyme SODs and... Transgenic and Knockout Mutant... Conclusion References

 
It is now clear that oxidants play a major role in brain damage in cerebrovascular diseases. The successful development of SOD-1 transgenic mice, as well as sod-1, sod-2, neuronal NOS knockout mutants, and many other transgenic and knockout mutant mice, has afforded stroke researchers and neuroscientists a unique opportunity to study the oxidant mechanisms underlying the complex neuronal responses to ischemic insults. It is clear that these genetically modified mice could also be useful for the study of neurodegenerative diseases and neurological disorders other than cerebrovascular diseases. Despite these successful beginnings, there are obvious advantages to using larger animal species (ie, rats) for transgenic animals and knockout mutants, since many models for stroke and cerebrovascular disease have been developed mainly in rats.⁵⁶ The successful creation of transgenic and knockout mutant rats will be an invaluable addition to the study of the mechanisms underlying ischemic brain damage in the well-established focal, thrombolytic, and global ischemia models.

	Selected Abbreviations and Acronyms

 

bFGF = basic fibroblast growth factor CCA = common carotid artery GSHPx = glutathione peroxidase H2O2 = hydrogen peroxide HO2· = perhydroxyl (radical) MCA = middle cerebral artery NO, NO· = nitric oxide, nitric oxide (radical) NOS = nitric oxide synthase O2·- = superoxide (radical) ·OH = hydroxyl (radical) ONOO- = peroxynitrite SOD = superoxide dismutase

	Acknowledgments

 
This work is supported by National Institutes of Health grants NS-14543, NS-25372, AG-08938, and NO1 NS-5-2334. I thank Cheryl Christensen for editorial assistance.

Received April 12, 1996; accepted April 19, 1996.

	References

Top Abstract Introduction Oxygen Radical Production in... Antioxidant Enzyme SODs and... Transgenic and Knockout Mutant... Conclusion References

 

1. Kontos HA. Oxygen radicals in cerebral vascular injury. Circ Res. 1985;57:508-516. [Abstract/Free Full Text]
2. Siesjö BK, Agardh CD, Bengtsson F. Free radicals and brain damage. Cerebrovasc Brain Metab Rev. 1989;1:165-211. [Medline] [Order article via Infotrieve]
3. Chan PH. Oxygen radicals in focal cerebral ischemia. Brain Pathol. 1994;4:59-65. [Medline] [Order article via Infotrieve]
4. Boveris A, Chance B. The mitochondrial generation of hydrogen peroxide. Biochem J. 1973;134:707-716. [Medline] [Order article via Infotrieve]
5. Oliver CN, Starke-Reed PE, Stadtman ER, Liu GJ, Carney JM, Floyd RA. Oxidative damage to brain proteins, loss of glutamine synthetase activity and production of free radicals during ischemia/reperfusion-induced injury to gerbil brain. Proc Natl Acad Sci U S A. 1990;87:5144-5147. [Abstract/Free Full Text]
6. Malinski T, Bailey F, Zhang ZG, Chopp M. Nitric oxide measured by a porphyrinic microsensor in rat brain after transient middle cerebral artery occlusion. J Cereb Blood Flow Metab. 1993;13:355-358. [Medline] [Order article via Infotrieve]
7. Tominaga T, Sato S, Ohnishi J, Ohnishi ST. Potentiation of nitric oxide formation following bilateral carotid occlusion and focal cerebral ischemia in the rat: in vivo detection of the nitric oxide radical by electron paramagnetic resonance spin trapping. Brain Res. 1993;614:342-346. [Medline] [Order article via Infotrieve]
8. Lafon-Cazal M, Pietri S, Culcasi M, Bockaert J. NMDA-dependent superoxide production and neurotoxicity. Nature. 1993;364:535-537. [Medline] [Order article via Infotrieve]
9. Dugan LL, Sensi SL, Canzoniero LMT, Handran SD, Rothman SM, Lin T-S, Goldberg MP, Choi DW. Mitochondrial production of reactive oxygen species in cortical neurons following exposure to N-methyl-D-aspartate. J Neurosci. 1995;15:6377-6388. [Abstract/Free Full Text]
10. Bindokas VP, Jordán J, Lee CC, Miller RJ. Superoxide production in rat hippocampal neurons: selective imaging with hydroethidine. J Neurosci. 1996;16:1324-1336. [Abstract/Free Full Text]
11. 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]
12. Dawson TM, Dawson VL, Snyder SH. A novel neuronal messenger molecule in brain: the free radical, nitric oxide. Ann Neurol. 1992;32:297-311. [Medline] [Order article via Infotrieve]
13. 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. [Abstract/Free Full Text]
14. Hue RT, Padmaja S. The reaction of NO with superoxide. Free Radic Res Commun. 1993;18:195-199. [Medline] [Order article via Infotrieve]
15. Beckman JS, Carson M, Smith CD, Koppenol WH. ALS, SOD and peroxynitrite. Nature. 1993;364:584. Letter. [Medline] [Order article via Infotrieve]
16. Iadecola C. Regulation of the cerebral microcirculation during neural activity: is nitric oxide the missing link? Trends Neurosci. 1993;16:206-214. [Medline] [Order article via Infotrieve]
17. Dawson VL, Dawson TM, Bartely DA, Uhl GR, Snyder SH. Mechanisms of nitric oxide-mediated neurotoxicity in primary brain cultures. J Neurosci. 1993;13:2651-2661. [Abstract]
18. Lipton SA, Choi YB, Pan ZH, Lei SZ, Chen HS, Sucher NJ, Loscalzo J, Singel DJ, Stamler JS. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds. Nature. 1993;364:626-632. [Medline] [Order article via Infotrieve]
19. Troy CM, Derossi D, Prochiantz A, Greene LA, Shelanski ML. Downregulation of Cu/Zn superoxide dismutase leads to cell death via the nitric oxide-peroxynitrite pathway. J Neurosci. 1996;16:253-261. [Abstract/Free Full Text]
20. Oberley LW. Superoxide Dismutase. Boca Raton, Fla: CRC Press; 1982;1:1-141.
21. Lieman-Hurwitz J, Dafni N, Lavie V, Groner Y. Human cytoplasmic superoxide dismutase cDNA clone: a probe for studying the molecular biology of Down syndrome. Proc Natl Acad Sci U S A. 1982;79:2808-2811. [Abstract/Free Full Text]
22. Marklund SL. Human copper-containing superoxide dismutase of high molecular weight. Proc Natl Acad Sci U S A. 1982;79:7634-7638. [Abstract/Free Full Text]
23. Chan PH, Epstein CJ, Kinouchi H, Imaizumi S, Carlson E, Chen SF. Role of superoxide dismutase in ischemic brain injury: reduction of edema and infarction in transgenic mice following focal cerebral ischemia. Prog Brain Res. 1993;96:97-104. [Medline] [Order article via Infotrieve]
24. He YY, Hsu CY, Ezrin AM, Miller MS. Polyethylene glycol-conjugated superoxide dismutase in focal cerebral ischemia-reperfusion. Am J Physiol. 1993;265:H252-H256. [Abstract/Free Full Text]
25. Chan PH, Longar S, Fishman RA. Protective effects of liposome-entrapped superoxide dismutase on post-traumatic brain edema. Ann Neurol. 1987;21:540-547. [Medline] [Order article via Infotrieve]
26. Imaizumi S, Woolworth V, Fishman RA, Chan PH. Liposome-entrapped superoxide dismutase reduces cerebral infarction in cerebral ischemia in rats. Stroke. 1990;21:1312-1317. [Abstract/Free Full Text]
27. Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci U S A. 1980;77:7380-7384. [Abstract/Free Full Text]
28. Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987;51:503-512. [Medline] [Order article via Infotrieve]
29. Kinouchi H, Epstein CJ, Mizui T, Carlson EJ, 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.[Abstract/Free Full Text]
30. 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]
31. 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]
32. Martinou JC, Dubois-Dauphin M, Staple JK, Rodriguez I, Frankowski H, Missotten M, Albertini P, Talabot D, Catsicas S, Pietra C. Overexpression of BCL-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia. Neuron. 1994;13:1017-1030. [Medline] [Order article via Infotrieve]
33. MacMillan V, Judge D, Wiseman A, Settle D, Swain J, Davis J. Mice expressing a bovine basic fibroblast growth factor transgene in the brain show increased resistance to hypoxemic-ischemic cerebral damage. Stroke. 1993;24:1735-1739. [Abstract/Free Full Text]
34. Mirochnitchenko O, Palnitkar U, Philbert M, Inouye M. Thermosensitive phenotype of transgenic mice overproducing human glutathione peroxidases. Proc Natl Acad Sci U S A. 1995;92:8120-8124. [Abstract/Free Full Text]
35. Huang Z, Huang PL, Panahian N, Dalkara T, Fishman M, Moskowitz MA. Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science. 1994;265:1883-1885. [Abstract/Free Full Text]
36. Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA, Fishman MC. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature. 1995;377:239-242. [Medline] [Order article via Infotrieve]
37. Huang Z, Huang PL, Fishman MC, Moskowitz MA. Focal cerebral ischemia in mice deficient in either endothelial (eNOS) or neuronal nitric oxide (nNOS) synthase. Stroke. 1996;27:173. Abstract.
38. MacMicking JD, Nathan C, Hom G, Chartrain N, Fletcher DS, Trumbauer M, Stevens K, Xie Q-W, Sokol K, Hutchinson N, Chen H, Mudgett JS. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell. 1995;81:641-650. [Medline] [Order article via Infotrieve]
39. Crumrine RC, Thomas AL, Morgan PF. Attenuation of p53 expression protects against focal ischemic damage in transgenic mice. J Cereb Blood Flow Metab. 1994;14:887-891. [Medline] [Order article via Infotrieve]
40. Kondo T, Reaume A, Huang TT, Carlson E, Chen S, Scott R, Epstein CJ, Chan PH. Target disruption of CuZn-superoxide dismutase gene in mice causes exacerbation of cerebral infarction and neurological deficits after focal cerebral ischemia and reperfusion. Soc Neurosci Abstr. 1995;21:1268. Abstract.
41. Mikawa S, Li Y, Huang TT, Carlson E, Chen S, Kondo T, Murakami K, Epstein CJ, Chan PH. Cerebral infarction is exacerbated in mitochondrial manganese superoxide dismutase (Sod-2) knockout mutant mice after focal cerebral ischemia and reperfusion. Soc Neurosci Abstr. 1995;21:1268. Abstract.
42. Epstein CJ, Avraham KB, Lovett M, Smith S, Elroy-Stein O, 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. [Abstract/Free Full Text]
43. Kamii H, Kinouchi H, Chen SF, Sharp FR, Epstein CJ, Chan PH. SOD-1 transgenic mice: an application to the study of ischemic brain injury. In: Ohnishi ST, ed. Membrane-Linked Diseases. Boca Raton, Fla: CRC Press; 1995;4:423-431.
44. Mattson MP, Lovell MA, Furukawa K, Markesbery WR. Neurotrophic factors attenuate glutamate-induced accumulation of peroxides, elevation of intracellular Ca²⁺ concentration, and neurotoxicity and increase antioxidant enzyme activities in hippocampal neurons. J Neurochem. 1995;65:1740-1751. [Medline] [Order article via Infotrieve]
45. Freese A, Finklestein SP, DiFiglia M. Basic fibroblast growth factor protects striatal neurons in vitro from NMDA-receptor mediated excitotoxicity. Brain Res. 1992;575:351-355. [Medline] [Order article via Infotrieve]
46. Yuan J, Shaham S, Ledoux S, Ellis HM, Horvitz HR. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. Cell. 1993;75:641-652. [Medline] [Order article via Infotrieve]
47. Kane DJ, Sarafian TA, Anton R, Hahn H, Gralla EB, Valentine JS, Ord T, Bredesen D. Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science. 1993;262:1274-1277. [Abstract/Free Full Text]
48. Greenlund LJ, Korsmeyer SJ, Johnson EM Jr. Role of BCL-2 in the survival and function of developing and mature sympathetic neurons. Neuron. 1995;15:649-661. [Medline] [Order article via Infotrieve]
49. Zhong LT, Sarafian T, Kane DJ, Charles AC, Mah SP, Edwards RH, Bredesen DE. bcl-2 inhibits death of central neural cells induced by multiple agents. Proc Natl Acad Sci U S A. 1993;90:4533-4537. [Abstract/Free Full Text]
50. Chen J, Graham SH, Chan PH, Lan J, Zhou RL, Simon RP. bcl-2 is expressed in neurons that survive focal ischemia in the rat. Neuroreport. 1995;6:394-398. [Medline] [Order article via Infotrieve]
51. Lawrence MS, Ho DY, Sun GH, Steinberg GK, Sapolsky RM. Overexpression of Bcl-2 with herpes simplex virus vectors protects CNS neurons against neurological insults in vitro and in vivo. J Neurosci. 1996;16:486-496. [Abstract/Free Full Text]
52. Huang PL, Dawson PM, Bredt DS, Snyder SH, Fishman ME. Targeted disruption of the neuronal nitric oxide synthase gene. Cell. 1993;75:1273-1286. [Medline] [Order article via Infotrieve]
53. Iadecola C, Xu X, Zhang F, El-Fakahany EE, Ross ME. Marked induction of calcium-independent nitric oxide synthase activity after focal cerebral ischemia. J Cereb Blood Flow Metab. 1995;15:52-59. [Medline] [Order article via Infotrieve]
54. Morrison RS, Wenzel HJ, Kinoshita Y, Robbins CA, Donehower LA, Schwartzkroin PA. Loss of the p53 tumor suppressor gene protects neurons from kainate-induced cell death. J Neurosci. 1996;16:1337-1345. [Abstract/Free Full Text]
55. Li Y, Huang T-T, Carlson EJ, Melov S, Ursell PC, Olson JL, Noble LJ, Yoshimura MP, Berger C, Chan PH, Wallace DC, Epstein CJ. Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. Nat Genet. 1995;11:376-381. [Medline] [Order article via Infotrieve]
56. Ginsberg MD. Models of cerebral ischemia in the rodent. In: Schurr A, Rigor BM, eds. Cerebral Ischemia and Resuscitation. Boca Raton, Fla: CRC Press; 1990:1-15.

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