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(Stroke. 2001;32:184.)
© 2001 American Heart Association, Inc.

Original Contributions

Gene Transfer of Extracellular Superoxide Dismutase Increases Superoxide Dismutase Activity in Cerebrospinal Fluid

Hiroshi Nakane, MD; Yi Chu, PhD; Frank M. Faraci, PhD; Larry W. Oberley, PhD Donald D. Heistad, MD

From the Departments of Internal Medicine and Pharmacology, Cardiovascular Center, and Radiation Research Laboratory (L.W.O.), University of Iowa College of Medicine, Iowa City.

Correspondence to Donald D. Heistad, MD, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242. E-mail donald-heistad{at}uiowa.edu

	Abstract

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Background and Purpose—Copper-zinc superoxide dismutase (CuZnSOD) is expressed intracellularly, while extracellular SOD (EC-SOD) is released from cells. The purpose of this study was to determine whether gene transfer of CuZnSOD increases SOD activity predominantly in tissues, and gene transfer of EC-SOD increases SOD activity in cerebrospinal fluid (CSF). We also determined whether heparin or dextran sulfate releases EC-SOD into CSF.

Methods—We injected recombinant adenoviruses expressing EC-SOD (AdEC-SOD), CuZnSOD (AdCuZnSOD), or ß-galactosidase (Adß-gal) into the cisterna magna of rabbits.

Results—Total SOD activity in CSF was 39±11 U/mL (mean±SE) before virus injection. Three days later, total SOD activity in CSF increased to 148±22 U/mL after AdEC-SOD and 92±10 U/mL after AdCuZnSOD (P<0.05 versus AdEC-SOD), with no change after Adß-gal (49±5 U/mL). EC-SOD protein was detected in CSF after AdEC-SOD but not AdCuZnSOD or Adß-gal. Injection of heparin or dextran sulfate into the cisterna magna increased total SOD activity 27-fold and 32-fold over basal values, respectively, in CSF of rabbits that received AdEC-SOD. In contrast to effects in CSF, total SOD activity in basilar artery and meninges was significantly higher after AdCuZnSOD and tended to be higher after AdEC-SOD than after Adß-gal.

Conclusions—We have developed a method for intracranial gene transfer of CuZnSOD and EC-SOD. After gene transfer, CuZnSOD was expressed mainly in tissues, and EC-SOD was released into the CSF, especially after injection of heparin or dextran sulfate. Gene transfer of different isoforms of SOD may be useful in studies of cerebral vascular physiology and pathophysiology.

Key Words: adenovirus • basilar artery • gene transfer • heparin • superoxide dismutase

	Introduction

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Superoxide may contribute to vascular dysfunction in several pathological states in the central nervous system, including ischemia-reperfusion injury,^{1 2 3} vasospasm after subarachnoid hemorrhage,^{4 5} and meningitis.⁶ Although there are several potent antioxidant systems, superoxide dismutase (SOD) appears to play a key role in protection against oxygen radicals.

There are 3 known isoforms of SOD: copper-zinc SOD (CuZnSOD), manganese SOD (MnSOD), and extracellular SOD (EC-SOD). CuZnSOD is an isoform of SOD that exists intracellularly and dismutes superoxide within cells.^{7 8} MnSOD is located in mitochondria and plays a critical role as an antioxidant.^{8 9} EC-SOD is the only isoform of SOD that is released from cells into the extracellular space.¹⁰ EC-SOD is the primary extracellular antioxidant enzyme and is highly expressed in blood vessels,^{11 12} uterus,¹³ and airways.¹² We speculated that, after intracranial gene transfer of CuZnSOD, CuZnSOD would remain within cells lining the subarachnoid space and SOD activity would increase within the cells. In contrast, we anticipated that after intracranial gene transfer of EC-SOD, EC-SOD would be produced within the cells and released into extracellular space. Thus, SOD activity might increase in both cells and cerebrospinal fluid (CSF).

Previous studies have reported that EC-SOD is released into plasma from liver after injection of heparin or dextran sulfate intravenously.¹⁴ The mechanism of this release is thought to be displacement of EC-SOD by heparin or dextran sulfate from heparan sulfate proteoglycan on cell surfaces.¹⁵ We speculated that heparin or dextran sulfate might release EC-SOD into CSF after transduction of EC-SOD to cells in the subarachnoid space.

Our goals were (1) to determine whether gene transfer of CuZnSOD and EC-SOD by injection into the cisterna magna increases SOD activity in cells lining the subarachnoid space and in CSF, (2) to determine whether the increase in SOD activity in CSF is greater after gene transfer of EC-SOD than CuZnSOD, and (3) to determine whether heparin or dextran sulfate enhances release of EC-SOD into the CSF.

	Materials and Methods

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Adenoviral Vectors
Three different recombinant adenoviruses were used for gene transfer: (1) AdCMVß-gal carried the reporter gene of ß-galactosidase and was used as a control virus; (2) AdCMVCuZnSOD, a gift from Dr John Engelhardt (University of Iowa), contained cDNA for human CuZnSOD; and (3) AdCMVEC-SOD carried cDNA for human extracellular SOD. The adenoviral vectors were produced by the University of Iowa Gene Transfer Vector Core with the method described previously.¹⁶ Phosphate-buffered saline (PBS) containing 3% sucrose, which is the solvent for viruses, was used as virus-free control.

In Vivo Gene Transfer to Rabbits
All animal procedures were approved by the Animal Care and Use Review Committee at the University of Iowa. Adult male New Zealand White rabbits (weight, 2.5 to 3.4 kg; n=34) were anesthetized with intramuscular ketamine 50 mg/kg and xylazine 10 mg/kg. A 25-gauge needle was aseptically inserted into the cisterna magna as previously described,¹⁷ Three hundred microliters of CSF was withdrawn for measurement of total SOD activity, and 300 µL of viral suspension (artificial CSF with Adß-gal, AdCuZnSOD, or AdEC-SOD in 3% sucrose) was injected slowly. The dose of adenovirus was 3x10⁹ plaque-forming units per injectate.

We observed the distribution of ß-gal expression in the brain of rabbits using X-gal staining after injection of AdCMVß-gal into the cisterna magna. We found that the ventral surface of the brain was extensively stained in rabbits (data not shown), as we had shown previously in rats,¹⁷ mice,¹⁸ and rabbits.¹⁹

One day after injection of virus suspension, rabbits were anesthetized with intramuscular ketamine 50 mg/kg and xylazine 10 mg/kg, and CSF was collected. Three days after injection, rabbits were killed by an overdose of pentobarbital. After collection of CSF, the basilar artery and meninges (dura) of the brain stem were obtained. Each CSF sample was centrifuged at 3000 rpm for 6 minutes to remove cell components. Supernatant was stored at -20°C until used. Basilar artery and meninges were frozen in liquid nitrogen and stored at -80°C until used.

In some rabbits, heparin (20 U/kg) or dextran sulfate (5000 molecular weight, 8 µg/kg) was injected into the cisterna magna 3 days after gene transfer of EC-SOD or CuZnSOD, and CSF samples were collected 15, 60, and 120 minutes after injection of heparin or dextran sulfate. We estimated the dose of heparin and dextran sulfate from a previous study, in which 2000 U/kg of heparin and 800 mg/kg of dextran sulfate were injected intravenously.¹⁴ Total blood volume is approximately 100x CSF volume, and we therefore injected a dose of heparin and dextran sulfate into CSF based on the ratio of the volume of blood and CSF. Total SOD activity was measured in the samples by the nitroblue tetrazolium (NBT) method.²⁰

Immunocytochemistry for EC-SOD
Unfixed frozen arteries were cut into 6-µm sections on a cryostat at -20°C. Sections were incubated with primary antibody (goat anti-human EC-SOD, 1:500) overnight. After they were washed with PBS, sections were incubated for 30 minutes with the secondary antibody (mouse anti-goat IgG, 1:1000), rinsed again with PBS, and developed with Vectastain ABC Kit alkaline phosphatase (Vector Laboratories Inc).

Total SOD Activity
Tissue Preparation
Transduced basilar arteries and meninges were homogenized with a tissue homogenizer (5-second bursts for 3 minutes) in 3 volumes of 0.05 mol/L phosphate buffer, pH 7.8, and sonicated with an ultrasonic processor (10-second bursts for 1 minute). All procedures were performed on ice. The homogenate was centrifuged at 3000 rpm for 10 minutes, and the supernatant was used for determination of protein content and total SOD activity.

SOD Assay
Total SOD activities of tissue and CSF were determined by the method of Spitz and Oberley.²⁰ Measurement of enzyme activity was based on inhibition of O₂^-–mediated reduction of NBT to formazan, the photoabsorbant product. Solutions containing 1 U/mL catalase, 5.6x10^-5 mol/L NBT, 0.1 mmol/L xanthine, 1.0 mmol/L diethylenetriaminepentaacetic acid, 0.13 mg/mL bovine serum albumin, and 50 µmol/L bathocuproine disulfonic acid in 0.05 mol/L phosphate buffer (pH 7.8) were incubated with varying amounts of supernatant (0 to 100 µg of protein in tissue samples, 0 to 100 µL in CSF samples). Superoxide radicals were generated by adding xanthine oxidase (10^-2 to 10^-3 U/mL) to the cuvettes, and measurements were recorded at 560 nm, 37°C for 5 minutes, with the use of a spectrophotometer. Total SOD activity of the supernatant was measured as the effectiveness of the supernatant to inhibit dismutation of O₂^- generated by the xanthine–xanthine oxidase reaction. Total SOD activities were expressed in units per milligram of protein measured, as described by Lowry et al,²¹ for tissue samples or units per milliliter for CSF samples.

Western Blotting for CuZnSOD and EC-SOD in CSF
To determine the isoforms of SOD in CSF, Western blotting was used. First, CSF samples were concentrated 10-fold by cryovaporization. CSF that contained 50 µg of protein was denatured with SDS loading buffer at 95°C for 5 minutes and then separated on an SDS/10% polyacrylamide gel with 5% stacking gel in SDS/Tris/glycine running buffer.²² We used a commercial CuZnSOD standard (Sigma) and made an EC-SOD standard using gene transfer with AdEC-SOD to C6 glioma cells. The protein was electrophoretically transferred to a nitrocellulose membrane, which was then blocked with 5% (wt/vol) nonfat milk in PBS containing 0.1% Tween-20 for 1 hour at room temperature. The membrane was then incubated with primary antibody, goat anti-human EC-SOD (1:5000), or sheep anti-human CuZnSOD (1:500) in PBS containing 0.1% Tween-20 for 1 hour. After it was washed, the blot was incubated with horseradish peroxidase–conjugated rabbit anti-goat IgG (1:10 000) or rat anti-sheep IgG (1:10 000) for 1 hour at room temperature. After it was washed, the blot was incubated with Super Signal ULTRA chemiluminescent substrate (Pierce Laboratories) and exposed to x-ray film.

Data Analysis
Results are expressed as mean±SE. Total SOD activity data in CSF, basilar arteries, and meninges were analyzed with 1-way ANOVA with post hoc Bonferroni correction.

	Results

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SOD in Tissue
Extensive expression of EC-SOD was observed in the adventitia of basilar artery from rabbits that were injected with AdEC-SOD. No EC-SOD staining was seen in basilar artery from rabbit that received Adß-gal (Figure 1). Endogenous EC-SOD of rabbit was not stained because the goat anti-human EC-SOD antibody did not detect rabbit EC-SOD.

View larger version (106K): [in this window] [in a new window]   Figure 1. Immunocytochemistry for EC-SOD in basilar artery from rabbits after injection of AdEC-SOD (B: magnification x128; D: x400) or Adß-gal (A: magnification x160; C: x400). Adv indicates adventitia. There was extensive blue staining for EC-SOD in adventitia of basilar artery after AdEC-SOD but not Adß-gal. No staining was observed in the absence of primary antibody. The sections were counterstained with nuclear fast red.

After injection of AdCuZnSOD into the cisterna magna, total SOD activity of basilar arteries was 2.5 times higher than that after injection of Adß-gal. Total SOD activity of basilar artery tended to increase after gene transfer of AdEC-SOD but was not significantly higher than that after Adß-gal (Figure 2).

View larger version (22K): [in this window] [in a new window]   Figure 2. Total SOD activity in basilar arteries and meninges 3 days after gene transfer of EC-SOD, CuZnSOD, or ß-gal. SOD activity in basilar arteries from rabbits that received AdCuZnSOD was significantly higher than after injection of Adß-gal. SOD activity in meninges from rabbits that received AdCuZnSOD was significantly higher than after injection of Adß-gal and AdEC-SOD. *P<0.01 vs Adß-gal. P<0.01 vs AdEC-SOD. Values are mean±SE; n=5 for Adß-gal, n=5 for AdEC-SOD, and n=6 for AdCuZnSOD.

Results were similar in the meninges. SOD activity in meninges was significantly higher 3 days after gene transfer of CuZnSOD than after EC-SOD or ß-gal (Figure 2).

SOD in CSF
There was no significant difference among the 3 groups in basal levels of total SOD activity in CSF. Three days after gene transfer of AdEC-SOD, total SOD activity was >3 times higher than basal level and was significantly higher than after injection of Adß-gal or AdCuZnSOD (Figure 3). Total SOD activity tended to increase after injection of AdCuZnSOD compared with Adß-gal, but this was not statistically significant.

View larger version (15K): [in this window] [in a new window]   Figure 3. Total SOD activity in CSF from rabbits that received AdEC-SOD, AdCuZnSOD, or Adß-gal. SOD activity in CSF after AdEC-SOD was significantly higher than after injection of Adß-gal or AdCuZnSOD. *P<0.01 vs Adß-gal. P<0.05 vs AdCuZnSOD. Values are mean±SE; n=6 for AdEC-SOD, n=6 for AdCuZnSOD, and n=5 for Adß-gal.

Western blotting of EC-SOD showed a band in CSF at the same position as the EC-SOD standard after injection of AdEC-SOD (Figure 4). This band was not detected in CSF samples from rabbits that were treated with AdCuZnSOD or Adß-gal. In contrast, CuZnSOD protein was undetectable in any samples of CSF. This result indicates that there is no detectable CuZnSOD protein in CSF even after gene transfer of CuZnSOD.

View larger version (36K): [in this window] [in a new window]   Figure 4. Western blotting of EC-SOD protein and CuZnSOD protein in CSF from rabbits 3 days after gene transfer of ß-gal (lane 1), CuZnSOD (lane 2), or EC-SOD (lane 3). EC-SOD protein was detected in CSF only from rabbits that received AdEC-SOD. CuZnSOD protein was not detected in any CSF samples, even after gene transfer of CuZnSOD. Lane 4 and 5 were EC-SOD standard and CuZnSOD standard, respectively.

Effects of Heparin and Dextran Sulfate
After transduction with AdEC-SOD, injection of heparin into the cisterna magna of rabbits increased total SOD activity by 6.7-fold, which was 27-fold the basal value for total SOD activity (Figure 5, left panel). The elevation was sustained for at least 2 hours after injection of heparin.

View larger version (13K): [in this window] [in a new window]   Figure 5. Total SOD activity in CSF from rabbits that received AdCuZnSOD or AdEC-SOD 3 days before these measurements. Left, After injection of heparin (20 U/kg) into the cisterna magna, total SOD activity increased in CSF of rabbits that received AdEC-SOD but not AdCuZnSOD. *P<0.01 vs AdCuZnSOD. Values are mean±SE; n=5 for AdEC-SOD, and n=5 for AdCuZnSOD. Right, After injection of dextran sulfate (5000 molecular weight, 8 µg/kg) into the cisterna magna, total SOD activity increased in CSF of rabbits that received AdEC-SOD but not AdCuZnSOD. *P<0.01 vs AdCuZnSOD. Values are mean±SE; n=4 for AdEC-SOD, and n=3 for AdCuZnSOD.

After transduction with AdEC-SOD, injection of dextran sulfate increased total SOD activity by 7.4-fold, which was a 33-fold increase above basal value in total SOD activity (Figure 5, right panel). Injection of heparin or dextran sulfate in rabbits that received AdCuZnSOD did not significantly increase total SOD activity in CSF.

	Discussion

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In this study we demonstrated that, first, overexpression of CuZnSOD and EC-SOD can be achieved in the subarachnoid space by in vivo gene transfer. Second, the distributions of CuZnSOD and EC-SOD are different after gene transfer. CuZnSOD is expressed mainly in cells after gene transfer and, in contrast, EC-SOD is released into the CSF. Third, injection of heparin or dextran sulfate into the cisterna magna greatly enhanced the release of EC-SOD into the CSF in rabbits that received AdEC-SOD.

Immunocytochemistry of EC-SOD showed extensive staining in the adventitia of basilar artery from rabbits that received AdEC-SOD. Furthermore, total SOD activity in basilar artery and meninges from rabbits that received AdCuZnSOD increased significantly compared with ß-gal transduced–rabbits. These results indicate that CuZnSOD and EC-SOD can be transduced in the basilar artery and meninges by injection of adenoviral vectors into the cisterna magna. In this study we used anti-human EC-SOD antibody, which detects human but not rabbit EC-SOD. Thus, we could detect the EC-SOD that is expressed by gene transfer by immunocytochemistry and Western blotting.

EC-SOD is the only isoform of SOD that is released into the extracellular space.¹⁰ Because the amount of EC-SOD in most organs is relatively small compared with CuZnSOD and MnSOD, it has been assumed that this enzyme does not play a major role in dismutation of superoxide in extracellular space.²³ However, recent studies indicate that in some tissues, such as blood vessels,^{11 12} uterus,¹³ and airways,¹² EC-SOD is the predominant isoform of SOD. Thus, effects of EC-SOD under physiological and pathological conditions are of interest. Recently, several studies using EC-SOD transgenic mice have reported that EC-SOD has neuroprotective effects²⁴ and anti-inflammatory effects.²⁵

EC-SOD has a heparin binding site and can bind to heparan sulfate proteoglycans on the cell surface. On the basis of affinity for heparin, EC-SOD is separated into type A (without affinity), type B (with weak affinity), and type C (with high affinity) for heparin.¹⁰ There are large interspecies differences in total plasma EC-SOD activity and affinity for heparin.²⁶ EC-SOD of rats is mainly present in the extracellular fluid because rats have no type C EC-SOD. On the other hand, as predicted from the affinity for heparin, EC-SOD of humans (type C) binds to the cell surface. In rabbits, type C is the predominant isoform of EC-SOD.²⁶

In this study we transduced human EC-SOD to the brain of rabbits. Thus, human EC-SOD was overexpressed in cells and released into the extracellular space and then presumably bound to extracellular matrix of tissues. Immunocytochemistry for EC-SOD demonstrated that human EC-SOD was expressed in the adventitia of basilar artery and meninges after gene transfer. Interestingly, and as anticipated, total SOD activity in CSF also increased after injection of AdEC-SOD. On the basis of the Western blotting, the increase of SOD activity was due to expression of human EC-SOD. There may be several explanations for the presence of human EC-SOD in CSF after gene transfer. First, although the majority of human EC-SOD is type C, type A and type B of EC-SOD may also be present.²⁶ Thus, these types of SOD, especially type B, may be released into the CSF. Second, the binding of type C EC-SOD for heparan sulfate proteoglycans on the cell surface appears to be rather loose compared with other proteins that bind to heparan sulfate proteoglycans.¹⁵ Thus, some amount of type C EC-SOD may be released into the CSF by heparin.

Injection of heparin or dextran sulfate into the cisterna magna produced a large increase of total SOD activity in CSF in rabbits after gene transfer of EC-SOD but not CuZn-SOD. Because total SOD activity is measured in CSF, the values represent both endogenous and exogenous SOD. Thus, the minimal increase in SOD activity shown in Figure 5, after heparin or dextran sulfate in rabbits that received AdCuZn-SOD, indicates that neither exogenous nor endogenous isoforms increased substantially. We speculate that heparin and dextran sulfate bind to the heparin binding site of EC-SOD and displace EC-SOD into CSF from cells lining the subarachnoid space. This result is consistent with a previous report that showed an increase in SOD activity in plasma after gene transfer of EC-SOD and injection of heparin or dextran sulfate intravenously.¹⁴

It is likely that, after injection of AdEC-SOD into the cisterna magna, some EC-SOD was released into the CSF, but the majority of the EC-SOD was bound to the extracellular matrix of tissues. Heparin and dextran sulfate then presumably displaced EC-SOD, which was bound to cells, from extracellular matrix into extracellular fluid. In a previous report, a 3-fold increase in CuZnSOD in neurons of transgenic mice had a protective effect against glutamate neurotoxicity.²⁷ In other reports, a 3- to 5-fold increase in EC-SOD in the brain or lung of transgenic mice had a protective effect against cerebral ischemia²⁴ or hyperoxic toxicity.²⁵ Thus, we anticipate that, although SOD activity in CSF is normally low, a 25- to 30-fold increase in SOD activity after gene transfer may be effective against oxidative stress produced by pathophysiological conditions such as subarachnoid hemorrhage, meningitis, or acute hypertension.

In contrast, gene transfer of CuZnSOD produced a substantial increase in SOD activity in tissues, with only a small increase in CSF. This finding is consistent with the concept that CuZnSOD is produced within cells and, after gene transfer, largely remains intracellular. However, total SOD activity in CSF tended to increase after gene transfer of CuZnSOD. One possible mechanism for the increase in SOD activity in CSF is that some cells, which were infected with AdCuZnSOD, may have undergone cell death, and endogenous rabbit SOD was released into the CSF. Because no human CuZnSOD protein was detected in CSF by Western blotting after gene transfer of CuZnSOD, it is unlikely that the elevation of SOD activity was produced by human SOD that was transduced to the rabbits.

As discussed above, it is most likely that the higher activity of SOD in CSF after gene transfer of EC-SOD than CuZnSOD is due to release of EC-SOD, but not CuZnSOD, from the extracellular compartment into the CSF. It is possible, however, that both SODs are released into CSF, and more rapid clearance of the smaller protein (CuZnSOD) contributes to low concentrations in CSF.

CuZnSOD may play an important role in protection against neuronal injury^{1 2 3} and vasospasm after subarachnoid hemorrhage.⁵ CuZnSOD improves vasomotor function^{28 29 30} by dismutation of superoxide anion within cells. Several reports have suggested that increases in intracellular CuZnSOD, using liposomal-encapsulated SOD^{31 32 33} or polyethylene glycol– conjugated SOD,^{29 30} are effective in protection of cells. Using gene transfer techniques, CuZnSOD can be overexpressed within cells. Thus, gene transfer of CuZnSOD also appears to be a promising approach to protect vessels from pathological conditions.

Efficiency of gene transfer varies among species and may be influenced by serotype of adenoviral vectors that is used for different species. Applications of this approach to different species, including humans, will require optimization of the vector, including perhaps the serotype of the adenovirus.

Studies using transgenic mice suggest that CuZnSOD and EC-SOD are key enzymes in protection against neuronal injury or vasomotor dysfunction.^{1 5 24 28} Furthermore, EC-SOD may be involved in regulation of vasomotor function³⁴ because there are substantial amounts of EC-SOD in blood vessels.^{11 12} We suggest that this intracranial in vivo gene transfer approach may be useful in clarifying the role of EC-SOD and CuZnSOD in cerebral vascular biology.

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-16066, HL-14388, NS-24621, HL-62984, and DK-54759. We thank Christine J. Darby for helpful advice regarding the SOD assay and Pamela K. Tompkins for assistance with immunocytochemistry. We thank Beverly Davidson and the Vector Core for providing the recombinant viruses and Dr James D. Crapo for providing the goat anti-human EC-SOD antibody.

Received April 25, 2000; revision received September 8, 2000; accepted September 8, 2000.

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Editorial Comment

Pak H. Chan, PhD, Guest Editor

Neurosurgical Laboratories, Stanford University, Stanford, California

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Oxygen free radicals, superoxide and nitric oxide in particular, are known to be involved in cerebral vascular signaling and injury after cerebral ischemia and reperfusion.^{R1 R2} Superoxide radicals are scavenged by superoxide dismutases (SODs), and 3 isoforms of SOD have been identified on the basis of their cellular localization. Both cytosolic copper-zinc SOD (CuZn SOD) and mitochondrial manganese SOD are localized intracellularly, whereas the third isoform, extracellular SOD (EC SOD), is localized mainly in the extracellular space.

Although transgenic or knockout mice of these SOD isoforms have been generated and used in stroke studies,^{R3 R4 R5 R6} it is not clear whether these enzymes can be expressed and upregulated by means other than the transgenic technology. In this nicely written and carefully done study, Nakane and his colleagues have injected an adenoviral vector with either the CuZn SOD or EC SOD gene into the cerebrospinal fluid and then looked at the distribution and expression pattern in brain tissue. The same group of investigators have used this gene transfer technique in vivo to cerebral blood vessels and perivascular tissue in mice.^R7 They have demonstrated that increased CuZn SOD activity was detected in brain tissue, whereas increased EC SOD activity was found only in the cerebrospinal fluid. Thus, the authors have successfully developed a method for intracranial gene transfer of CuZn SOD and EC SOD that can target the intracellular or extracellular compartments, respectively. In view of the importance of oxidative stress in cerebral vascular responses after cerebral ischemia and reperfusion, this method of gene transfer of different isoforms of SOD at different cellular locations is extremely useful to dissect out the divergent roles of these isoforms in signaling and neuroprotection in ischemic brain damage.

Received April 25, 2000; revision received September 8, 2000; accepted September 8, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 

1. Kontos HA. George E. Brown Memorial Lecture: oxygen radicals in cerebral vascular injury. Circ Res. 1985;57:508–516.[Abstract/Free Full Text]
2. Chan PH. Role of oxidants in ischemic brain damage. Stroke. 1996;27:1124–1129.[Abstract/Free Full Text]
3. 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.
4. Kondo T, Reaume AG, Huang T-T, 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.[Abstract/Free Full Text]
5. 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.[Abstract/Free Full Text]
6. 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.
7. Christenson SD, Lake KD, Ooboshi H, Faraci FM, Davidson BL, Heistad DD. Adenovirus-mediated gene transfer in vivo to cerebral blood vessels and perivascular tissue in mice. Stroke. 1998;29:1411–1416.

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