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(Stroke. 2003;34:434.)
© 2003 American Heart Association, Inc.

Original Contributions

Gene Transfer of Extracellular Superoxide Dismutase Reduces Cerebral Vasospasm After Subarachnoid Hemorrhage

Yoshimasa Watanabe, MD; Yi Chu, PhD; Jon J. Andresen, BA; Hiroshi Nakane, MD; Frank M. Faraci, PhD Donald D. Heistad, MD

From the Departments of Internal Medicine and Pharmacology, Cardiovascular Center, University of Iowa College of Medicine and Veterans Affairs Medical Center, Iowa City, Iowa.

Correspondence to Donald D. Heistad, MD, Department of Internal Medicine, University of Iowa College of Medicine, 200 Hawkins Dr, Iowa City, IA 52242. E-mail donald-heistad{at}uiowa.edu

	Abstract

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Background and Purpose— Superoxide may play an important role in cerebral vasospasm after subarachnoid hemorrhage (SAH). Our first goal was to determine the effect of gene transfer of extracellular superoxide dismutase (ECSOD) on vasospasm after experimental SAH. Our second goal was to determine whether tissue binding of ECSOD via the heparin-binding domain (HBD) is important for the effect of the enzyme. Thus, we examined effects of gene transfer of ECSOD with the HBD deleted (ECSODHBD).

Methods— Adenovirus expressing human ECSOD (AdECSOD), ECSODHBD (AdECSODHBD), or no transgene (AdBglII) was injected into the cisterna magna of anesthetized rabbits 30 minutes after induction of experimental SAH. Cerebral angiography, an assay for ECSOD activity in cerebrospinal fluid (CSF), and Western blotting for human ECSOD in the basilar artery were performed.

Results— Baseline diameter of the basilar artery averaged 0.77±0.01 mm (mean±SEM) and was similar in all treatment groups. Decreases in diameter of the basilar artery 2 days after SAH were smaller after AdECSOD (11±3%; n=10) than after AdBglII (25±4%; n=7; P<0.05). ECSOD activity was not detected in CSF before SAH and gene transfer. Of 8 rabbits treated with AdECSOD, in which ECSOD activity in CSF was measured after SAH, 5 animals had detectable ECSOD activity in CSF (46±13 U/mL). In these 5 rabbits, the diameter decreased by only 6±3%, and ECSOD protein was detected in the basilar artery. After AdECSODHBD (n=4), despite high levels of ECSOD activity in CSF (91±19 U/mL), vessel diameter decreased by 20±2%, and no ECSODHBD protein was detected in the basilar artery.

Conclusions— Gene transfer of ECSOD reduces cerebral vasospasm after experimental SAH. Tissue binding of the enzyme is essential for cerebral vascular effects of ECSOD.

Key Words: gene transfer • subarachnoid hemorrhage • superoxide dismutase • vasospasm, intracranial • rabbits

	Introduction

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Delayed cerebral vasospasm after subarachnoid hemorrhage (SAH) may be caused by multiple and complex mechanisms, including vascular dysfunction caused by oxidative stress.^1,2 Several studies indicate a pathophysiological role of superoxide (O₂^·-) in cerebral vasospasm. After experimental SAH in dogs, production of O₂^·- is increased in the subarachnoid space.³ Administration of superoxide dismutase (SOD) into cerebrospinal fluid (CSF) reduces cerebral vasospasm in rabbits,⁴ although this is not consistent in different animal models and treatment protocols.^5,6 In studies using transgenic mice that overexpress copper/zinc SOD (Cu/ZnSOD) or extracellular SOD (ECSOD), diameter of the middle cerebral artery after experimental SAH is larger in transgenic mice than in nontransgenic mice.^7,8 Some of these studies imply that production of O₂^·- in the extracellular space after SAH is important in the pathophysiology of vasospasm. Thus, we speculated that local overexpression of ECSOD, the only isoform of SOD that is secreted into the extracellular space,⁹ in CSF or cerebral arteries may be a useful approach to reduce vasospasm. The first goal of this study was to determine effects of gene transfer of ECSOD on cerebral vasospasm after experimental SAH.

ECSOD binds to tissue via its heparin-binding domain (HBD), which provides affinity of the enzyme for heparan-sulfate proteoglycans on cell surfaces and in extracellular matrices.¹⁰ ECSOD is released into blood by intravenous injection of heparin.¹¹ After gene transfer into the cisterna magna of normal rabbits, ECSOD is overexpressed in cerebral arteries and CSF, with the majority being bound to tissue and released into CSF by intracisternal heparin.¹² Distribution of overexpressed ECSOD may be a critical determinant of efficacy of gene transfer because there are at least 2 different possible sources of extracellular O₂^·-, oxyhemoglobin in CSF¹³ and NAD(P)H oxidase in cerebral arteries,¹⁴ that may contribute to the pathophysiology of vasospasm after SAH. Recently, we have constructed a human ECSOD with the HBD deleted (ECSODHBD).¹⁵ This variant of ECSOD, which has normal activity but does not have affinity for heparin or heparan-sulfate proteoglycans,¹⁰ is readily released into blood after systemic gene transfer in rats. We speculated that ECSODHBD would be released freely into CSF after gene transfer into the cisterna magna and that loss of tissue binding might change the efficacy of the enzyme for reduction of vasospasm. Thus, the second goal of this study was to compare effects of gene transfer of ECSOD and ECSODHBD after SAH.

	Materials and Methods

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Adenoviral Vectors
We constructed replication-deficient adenoviruses containing the gene for human ECSOD (AdECSOD) or human ECSODHBD (AdECSODHBD) with a cytomegalovirus promoter, using standard methods.¹⁶ An adenovirus with no transgene (AdBglII) was used as a control virus.

Gene Transfer in Rabbits Without SAH
All animal procedures were approved by the Animal Care and Use Review Committee of the University of Iowa.

Changes in total SOD activity in CSF were compared after gene transfer of ECSOD and ECSODHBD in rabbits without SAH. A method for gene transfer into the cisterna magna of the rabbit was described elsewhere.¹⁷ In brief, a recombinant adenovirus in phosphate-buffered saline (PBS) with 3% sucrose was diluted to 0.3 mL of viral suspension containing 1x10¹¹ particles with artificial CSF (composition in mmol/L: 132 NaCl, 2.95 KCl, 1.71 CaCl₂, 0.65 MgCl₂, 24.6 NaHCO₃, and 3.69 D-glucose). The titer of adenovirus was regarded to be optimal on the basis of previous experiments.¹² Adult male New Zealand White rabbits weighing 2.6 to 3.7 kg were anesthetized with intramuscular xylazine 10 mg/kg and ketamine 50 mg/kg, and a 25-gauge needle was aseptically inserted into the cisterna magna. After withdrawal of 0.5 mL of CSF, the viral suspension was slowly injected into the cisterna magna. The head was tilted nose-down by 30 degrees for 30 minutes after injection of virus.

One, 2, and 3 days after gene transfer, CSF samples were collected from the cisterna magna of anesthetized rabbits. Three days after gene transfer, a CSF sample was collected again 60 minutes after injection of heparin (20 U/kg) into the cisterna magna as described previously.¹² CSF samples were centrifuged at 1000g for 6 minutes to remove cell components and stored at -20°C until used. Total SOD activity in each CSF sample was measured by the modified nitroblue tetrazolium reduction method¹⁸ as described previously.¹²

Simulated SAH and Gene Transfer
Experimental SAH was produced in rabbits as described previously.¹⁹ In brief, a 25-gauge needle was aseptically inserted into the cisterna magna of an anesthetized rabbit, and 0.8 mL/kg of fresh nonheparinized autologous blood taken from the ear artery was injected after withdrawal of 0.8 mL of CSF. The head was tilted nose-down by 30 degrees for 30 minutes after injection of blood.

About 40 minutes after injection of blood, the viral suspension was slowly injected into the cisterna magna through a 25-gauge needle. Then the head was tilted nose-down by 30 degrees for 30 minutes.

Vertebral Angiography
Digital subtraction angiography was performed immediately before (day 0) and 2 days after (day 2) experimental SAH as previously described.¹⁹ In brief, anesthetized rabbits were placed in a supine position and mechanically ventilated. An angiogram was obtained by injection of 0.8 mL of nonionic contrast medium through a microcatheter (Renegade 130/20/1TIP; Boston Scientific) introduced into the left vertebral artery from an exposed femoral artery. Arterial pressure at the thoracic aorta was recorded directly through the catheter, and arterial blood gases were measured during angiography. Angiograms were converted to image files on a computer. Diameter of the basilar artery was measured with the NIH Image program by an observer blinded to the experimental protocol.

ECSOD Activity in CSF After SAH
CSF samples were collected from rabbits on day 2 after SAH, centrifuged, and stored at -20°C until used. To eliminate contamination of SOD activity from erythrocytes in the subarachnoid space, ECSOD and ECSODHBD were separated from other 2 isoforms, Cu/ZnSOD and manganese SOD (MnSOD), with affinity column chromatography on concanavalin A.²⁰ Each sample (0.3 mL) was applied to a 2-mL disposable column containing 0.5 mL of concanavalin A sepharose (Sigma). Cu/ZnSOD and MnSOD lack affinity for concanavalin A and were collected in the effluent from 1.5 mL of PBS added on the column. After the column was washed with 10 mL of PBS, ECSOD was eluted in 5 fractions of 0.3-mL effluent by addition of 0.5 mol/L -methyl-D-mannoside (Sigma) dissolved in PBS. SOD activity in each fraction containing ECSOD was determined by the nitroblue tetrazolium method and summed for each original sample. We estimated the recovery rate of SOD activity after the chromatography procedure as 60% for both ECSOD and ECSODHBD, using control enzymes made by gene transfer to A549 cells with AdECSOD or AdECSODHBD, respectively. Values of ECSOD activity in this study were corrected for this recovery rate. An activity of <10 U/mL is undetectable by the SOD activity assay used in this study, and therefore the lower detectable limit of ECSOD activity was calculated as 17 U/mL. In some animals, ECSOD activity also was measured in CSF samples withdrawn before induction of experimental SAH.

Western Blotting for ECSOD in Basilar Artery
In preliminary experiments, activity of ECSOD in the basilar artery was not measurable because the tissue sample was too small. Thus, overexpression of human ECSOD in the basilar artery was confirmed by Western blotting. Rabbits were euthanatized by an overdose of pentobarbital after angiography and after CSF samples were obtained on day 2. The basilar artery was removed, frozen in liquid nitrogen, and stored at -80°C until use. The frozen sample was ground into a coarse powder state, 100 µL of 0.05-mmol/L phosphate buffer (pH 7.8) was added, and protein extract was collected as a supernatant after centrifugation at 4°C and 15 000g for 15 minutes. Concentration of protein was determined by Lowry assay (Bio-Rad). Ten micrograms of protein was electrophoresed in a 10% native polyacrylamide gel and immunoblotted with a rabbit anti-ECSOD antibody (provided by Dr James Crapo of National Jewish Medical and Research Center, Denver, Colo). The blot was incubated with chemiluminescent substrate (Femto Maximum Sensitivity; Pierce) and exposed to x-ray film.

Data Analysis
Values are expressed as mean±SEM. Paired or unpaired t test was used for comparison of 2 values. Time-dependent changes in total SOD activity in CSF were analyzed by repeated-measures ANOVA and contrast analysis. One-way ANOVA followed by Scheffé’s F test was used for multiple comparisons. Mann-Whitney U test or Kruskal-Wallis test followed by Scheffé’s F test was used for comparison of ECSOD activity in CSF. P<0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Gene Transfer in Rabbits Without SAH
All rabbits without SAH tolerated an injection of AdECSOD (n=3) or AdECSODHBD (n=3) into the cisterna magna. In both groups total SOD activity in CSF increased during the 3 days after gene transfer (P<0.05), and the values at 2 and 3 days after gene transfer tended to be higher in AdECSODHBD-injected rabbits than in AdECSOD-injected animals (Figure 1A). Three days after gene transfer of ECSOD, total SOD activity in CSF was greatly increased by intracisternal injection of heparin (Figure 1B). In contrast, total SOD activity in CSF was not changed by heparin after gene transfer of ECSODHBD.

View larger version (16K): [in this window] [in a new window]   Figure 1. Changes in total SOD activity in CSF of rabbits without SAH after gene transfer of ECSOD or ECSODHBD into the cisterna magna. A, Total SOD activity increased in both AdECSOD-treated (n=3) and AdECSODHBD-treated (n=3) animals (P<0.05). *P<0.01 vs before gene transfer. B, Effect of an intracisternal injection of heparin on total SOD activity in CSF. Three days after gene transfer, CSF samples were collected before and 60 minutes after heparin. *P<0.05 vs before heparin. Values are mean±SEM.

Experimental SAH
Experimental SAH was induced in 36 rabbits (14 for AdBglII, 14 for AdECSOD, and 8 for AdECSODHBD group). Of these rabbits, 21 animals (7 for AdBglII, 10 for AdECSOD, and 4 for AdECSODHBD group) had subarachnoid clot covering the ventral surface of the brain stem. The other animals, which died (n=1) or had severe neurological deficits (n=5) immediately after the experimental procedures on day 0 or had only a small amount of blood in the subarachnoid space on postmortem examination (n=9), were excluded from further analyses.

Mean arterial blood pressure and arterial blood PCO₂ during angiography were not significantly different between day 0 and 2 in each group. There were small changes in arterial PO₂ and pH in some groups (Table).

View this table: [in this window] [in a new window]   Arterial Pressure and Blood Gases During Angiography in Rabbits Treated With AdBgIII, AdECSOD, or AdECSODHBD After Subarachnoid Hemorrhage

Effect of Gene Transfer of ECSOD on Vasospasm
Baseline diameter of the basilar artery was similar in the AdBglII and AdECSOD groups. In both groups, the diameter was decreased on day 2 after SAH (Figure 2A). The decrease in the diameter was smaller in the AdECSOD group than in the AdBglII group (Figure 2B).

View larger version (14K): [in this window] [in a new window]   Figure 2. Changes in diameter of basilar artery after experimental SAH in rabbits. Cerebral angiography was performed before (day 0) and 2 days after (day 2) SAH. A, Arterial diameters on day 0 and day 2. Rabbits were treated with AdECSOD (n=10) or AdBglII (n=7) approximately 40 minutes after SAH. *P<0.05 vs day 0. B, Percent changes in diameter on day 2 compared with day 0. *P<0.05 vs AdBglII group. Values are mean±SEM.

ECSOD Activity in CSF After Gene Transfer of ECSOD
ECSOD activity was not detected in CSF obtained before experimental SAH and gene transfer (n=7). On day 2, a CSF sample was measured for ECSOD activity from 5 rabbits of the AdBglII group and 8 animals of the AdECSOD group. In rabbits treated with AdBglII, ECSOD activity was not detected in CSF except for 1 animal that had 18 U/mL of activity in CSF, while in AdECSOD-treated rabbits it was detected in 5 animals (46±13 U/mL) except for 3 with no detectable activity. ECSOD activity was significantly higher in rabbits treated with AdECSOD than in animals treated with AdBglII (P<0.05).

To determine whether attenuation of the decrease in diameter depends on ECSOD activity in CSF after gene transfer of ECSOD, data from the rabbits described above were plotted against ECSOD activity (Figure 3A). Of 8 rabbits treated with AdECSOD, the percent decrease in the diameter was smaller in 5 animals that had detectable ECSOD activity in CSF. These 5 rabbits and the other 3 with no detectable ECSOD activity were separated into 2 subgroups: the AdECSOD[act(+)] and AdECSOD[act(-)] groups, respectively (Figure 4A). In the AdECSOD[act(+)] group, the diameter decreased by only 6±3% (P<0.05 versus the AdBglII and AdECSOD[act(-)] group) (Figure 4B).

View larger version (13K): [in this window] [in a new window]   Figure 3. Relationships between ECSOD activity in CSF and change in diameter of basilar artery after SAH. A, Data for change in diameter from rabbits treated with AdBglII or AdECSOD were plotted against ECSOD activity. B, Data from rabbits treated with AdECSODHBD (n=4) or AdECSOD were plotted. ND indicates not detectable.

View larger version (18K): [in this window] [in a new window]   Figure 4. Effect of gene transfer of ECSOD with or without the HBD on ECSOD activity in CSF and change in diameter of basilar artery after SAH. A, ECSOD activity of individual rabbits shown in Figure 3. Eight rabbits treated with AdECSOD were divided into 2 subgroups according to ECSOD activity in CSF: AdECSOD[act(+)] and AdECSOD[act(-)] indicate rabbits with and without detectable ECSOD activity in CSF after treatment with AdECSOD, respectively. B, Change in diameter of each group in A. Values are mean±SEM. *P<0.05 vs AdBglII, AdECSOD[act(-)], or AdECSODHBD group.

Effect of Gene Transfer of ECSODHBD
In all rabbits treated with AdECSODHBD, ECSOD activity in CSF after SAH was detectable (n=4). Baseline diameter of the basilar artery (0.80±0.03 mm; n=4) was similar to the AdBglII and AdECSOD groups. In contrast to rabbits treated with AdECSOD, there was no reduction in the severity of vasoconstriction, despite a large increase in ECSOD activity in CSF, in AdECSODHBD-treated animals (Figure 3B). The decrease in diameter in the AdECSODHBD group was comparable to that in the AdECSOD[act(-)] group or in the AdBglII group (Figure 4B).

Overexpression of Human ECSOD Protein in Basilar Artery
Binding of ECSOD to the basilar artery after treatment with AdECSOD, but not AdECSODHBD, was confirmed by Western blotting with the use of anti-human ECSOD antibody (Figure 5). After treatment with AdECSOD, only a small amount of human ECSOD protein was detected in the basilar artery of rabbits with no detectable ECSOD activity in CSF, while a large amount was detected in rabbits with elevated activity in CSF. No human ECSOD protein was detected in the basilar artery of rabbits treated with AdBglII or AdECSODHBD.

View larger version (114K): [in this window] [in a new window]   Figure 5. Detection of human ECSOD protein in basilar artery of a rabbit treated with AdBglII, AdECSOD, or AdECSODHBD after SAH. Results of Western blotting using anti-human ECSOD antibody are shown. Similar results were obtained from 6 other rabbits. Right 2 lanes are controls for ECSOD or ECSODHBD made by gene transfer of A549 cells with AdECSOD or AdECSODHBD, respectively. In most blots, ECSOD protein is detected as a smear rather than a band.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study indicates that gene transfer of ECSOD reduces cerebral vasospasm after experimental SAH. The decrease in arterial diameter was greatly reduced in AdECSOD-treated rabbits with increased ECSOD activity in CSF. In contrast, gene transfer of ECSODHBD failed to reduce vasospasm, despite a large increase in ECSOD activity in CSF. After gene transfer, a large amount of ECSOD, but no ECSODHBD, was bound to the basilar artery. These results suggest that tissue-bound ECSOD is essential for reduction of vasospasm after SAH.

Role of Superoxide in Vasospasm
Attenuation of cerebral vasospasm by exogenous SOD,⁴ in ECSOD transgenic mice,⁸ and by gene transfer of ECSOD (this study) suggests that O₂^·- produced in the extracellular space plays an important role in the pathophysiology of vasospasm. In preliminary experiments, we could not demonstrate an increase in O₂^·- level in the basilar artery after SAH either by lucigenin-enhanced chemiluminescence or by laser confocal microscopy using dihydroethidium (data not shown). We had anticipated that the lucigenin-enhanced signal would be increased by extracellular O₂^·-, whereas intracellular O₂^·- detected by dihydroethidium would not be increased. However, an increase in extracellular O₂^·- production after experimental SAH has been demonstrated previously in situ, in the subarachnoid space and in adventitia of the basilar artery.³

Extracellular O₂^·- may contribute to cerebral vasospasm after SAH through several mechanisms. O₂^·- inactivates nitric oxide (NO) to impair NO-mediated vasorelaxation.^21,22 Reaction of O₂^·- with NO produces the highly reactive oxidant peroxynitrite, which can alter protein function by nitration of tyrosine residues,²³ damage cell membranes by lipid peroxidation,²⁴ and break DNA.²⁵ There is evidence for possible involvement of these mechanisms in development of cerebral vasospasm.^8,26,27

Increased production of O₂^·- may result in elevation of levels in cerebral arteries of hydrogen peroxide (H₂O₂), which induces constriction in isolated basilar arteries.²⁸ Hydrogen peroxide can generate highly reactive hydroxyl radical in the presence of ferrous ion that may be increased after SAH,³ and studies using scavengers of hydroxyl radical indicate a role for this radical in vasospasm.²⁹ In vascular tissue, H₂O₂ can also activate key signaling molecules such as protein kinase C, mitogen-activated protein kinases, and nuclear factor-B,³⁰ which may be implicated in vasospasm after SAH.^31–33 Gene transfer of ECSOD could enhance these H₂O₂-mediated mechanisms by facilitating dismutation of O₂^·- to H₂O₂. However, 1 mol or more of H₂O₂ can be formed per mole of O₂^·- through oxidation of biomolecules such as catecholamines, whereas only 0.5 mol of H₂O₂ is produced by dismutation of 1 mol of O₂^·-. Thus, when production of O₂·- is enhanced in the subarachnoid space, efficient dismutation of O₂^·- by ECSOD may decrease levels of H₂O₂ by preventing stoichiometrically greater formation of H₂O₂ through the oxidizing action of O₂^·-.³⁴

Importance of Local Overexpression of ECSOD in Basilar Artery
Overexpression of ECSOD is achieved mainly in adventitia of cerebral arteries and meninges after in vivo gene transfer.¹² Thus, we speculate that vasospasm was attenuated by dismutation of extracellular superoxide in adventitia of the basilar artery, where ECSOD accumulates after gene transfer. Reduction of tyrosine nitration in adventitia of the middle cerebral artery after SAH in ECSOD transgenic mice provides support for this possibility.⁸ The half-life of O₂^·- at physiological pH is short so that O₂^·- does not diffuse far from its production site. Thus, it is possible that O₂^·-, which contributes to vasospasm after SAH, may be produced within adventitia of cerebral arteries. There are at least 2 possible sources of extracellular O₂^·- that may contribute to the pathophysiology of vasospasm after SAH. First, oxyhemoglobin, which is released from erythrocytes in the subarachnoid blood surrounding cerebral arteries, can release O₂^·- through autoxidation.¹³ Second, NAD(P)H oxidase in cerebral artery may be activated after SAH and contribute to development of vasospasm.¹⁴ NAD(P)H oxidase activity may be attributable to inflammatory cells infiltrating into cerebral arteries and perivascular tissue after SAH.³⁵ Adventitial fibroblasts also have components of NAD(P)H oxidase and produce extracellular O₂^·- in aorta,²² although it is not known whether a similar mechanism is present in intracranial arteries.

We suggest that tissue binding of ECSOD, from local overexpression of the enzyme in the basilar artery after gene transfer, is essential for the effect of the enzyme on vasospasm after SAH. For reduction of vasospasm by administration of exogenous SOD,⁴ repetitive intracisternal injections of an extremely large amount of Cu/ZnSOD is required to maintain adequate concentration of the enzyme in cerebral arteries, presumably because it does not bind to tissue.

ECSOD Activity in CSF After Gene Transfer
Absolute values of SOD activity in CSF were lower in rabbits after SAH than in rabbits without SAH, mainly because we measured ECSOD activity after SAH and total SOD activity in rabbits without SAH. It is known, however, that SOD activity in CSF is decreased after SAH.²⁶ Nevertheless, overexpression of ECSOD achieved in this study was sufficient for pronounced reduction of vasospasm. Increased ECSOD activity in CSF after gene transfer is associated with a large amount of ECSOD bound to the basilar artery (Figure 5). Only a small amount of ECSOD was overexpressed in 3 rabbits treated with AdECSOD, perhaps because we failed to successfully inject the virus into CSF through the cisterna magna.

Clinical Implications
In the rabbit model of SAH, vasospasm usually peaks approximately 2 days after injection of blood and resolves within 10 days.^4,36 After injection of AdECSOD into CSF of rabbits, the time course of overexpression of ECSOD coincides with the onset of vasospasm (Figure 1). A single injection of AdECSOD provides continuous production and accumulation of ECSOD in CSF and cerebral arteries for several days and was effective in reducing vasospasm. Thus, adenovirus-mediated gene transfer of ECSOD might potentially be useful as an antioxidant therapy for prevention of cerebral vasospasm after SAH. Previously, we reported that gene transfer of CGRP after induction of experimental SAH reduced vasospasm.¹⁹ Although CGRP may be more effective than ECSOD, gene therapy with ECSOD also is attractive because it may eliminate one of the possible causes of vasospasm after SAH.

In a large clinical trial, pegorgotein (polyethylene glycol–conjugated Cu/ZnSOD) failed to improve neurological deficits after head injury in patients.³⁷ There are many possible explanations for the finding, including the dose of SOD, timing and duration of treatment, and severity and type of injury. We would like to speculate about another possibility. Perhaps extracellular binding to the outer cell membrane is necessary for a protective effect of SOD. If extracellular O₂^·- contributes to secondary damage after head injury, our data imply that ECSOD might be beneficial only if it has the HBD, and Cu/ZnSOD, which does not have the HBD, would not be protective.

Limitations of the Study
We recognize that injection of blood into CSF of rabbits is not equivalent to SAH in patients. Rabbits develop relatively mild vasospasm (20% to 30% reduction in diameter in most studies),^4,19,26,33 which usually does not produce neurological deficits, with a short interval after onset of SAH, compared with patients. Further studies using canine or primate models of vasospasm, with comparable severity and temporal profile to patients, are needed to address this limitation.³⁶

Because inflammation may contribute to vasospasm after SAH,³⁵ inflammatory response to adenoviral vectors in CSF¹⁷ may have complicated the results of this study. To address this concern, we injected AdBglII as a control virus and compared effects of AdECSOD and AdBglII. In addition, previous experiments indicate that vasoconstriction after SAH is similar after SAH alone and SAH with a control virus.¹⁹ Inflammatory response to viral vectors will be one of the major obstacles to effective gene therapy for vasospasm after SAH. Further progress in gene transfer vectors is needed to overcome this problem.³⁸

	Acknowledgments

 
This work was supported by funds from the Veterans Administration; National Institutes of Health grants HL16066, HL62984, NS24621, and HL14388; an award from the American Heart Association (0120641Z); Carver Research Program of Excellence; and the Wendy Hamilton Trust. We thank Dr James Crapo of National Jewish Medical and Research Center, Denver, Colo, for providing human ECSOD cDNA plasmid and an antibody against ECSOD; Michael Ryan; and Jacque Wertz for assistance with angiography. We acknowledge the University of Iowa Gene Transfer Vector Core (and DK54759) for viral vector preparations.

Received May 20, 2002; revision received July 31, 2002; accepted August 28, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Sobey CG, Faraci FM. Subarachnoid haemorrhage: what happens to the cerebral arteries? Clin Exp Pharmacol Physiol. 1998; 25: 867–876.[Medline] [Order article via Infotrieve]
2. Dietrich HH, Dacey RG Jr. Molecular keys to the problems of cerebral vasospasm. Neurosurgery. 2000; 46: 517–530.[Medline] [Order article via Infotrieve]
3. Mori T, Nagata K, Town T, Tan J, Matsui T, Asano T. Intracisternal increase of superoxide anion production in a canine subarachnoid hemorrhage model. Stroke. 2001; 32: 636–642.[Abstract/Free Full Text]
4. Shishido T, Suzuki R, Qian L, Hirakawa K. The role of superoxide anions in the pathogenesis of cerebral vasospasm. Stroke. 1994; 25: 864–868.[Abstract]
5. Kajita Y, Suzuki Y, Oyama H, Tanazawa T, Takayasu M, Shibuya M, Sugita K. Combined effect of L-arginine and superoxide dismutase on the spastic basilar artery after subarachnoid hemorrhage in dogs. J Neurosurg. 1994; 80: 476–483.[Medline] [Order article via Infotrieve]
6. Macdonald RL, Weir BKA, Runzer TD, Grace MGA, Poznansky MJ. Effect of intrathecal superoxide dismutase and catalase on oxyhemoglobin-induced vasospasm in monkeys. Neurosurgery. 1992; 30: 529–539.[Medline] [Order article via Infotrieve]
7. Kamii H, Kato I, Kinouchi H, Chan PH, Epstein CJ, Akabane A, Okamoto H, Yoshimoto T. Amelioration of vasospasm after subarachnoid hemorrhage in transgenic mice overexpressing CuZn-superoxide dismutase. Stroke. 1999; 30: 867–872.[Abstract/Free Full Text]
8. McGirt MJ, Parra A, Sheng H, Higuchi Y, Oury TD, Laskowitz DT, Pearlstein RD, Warner DS. Attenuation of cerebral vasospasm after subarachnoid hemorrhage in mice overexpressing extracellular superoxide dismutase. Stroke. 2002; 33: 2317–2323.[Abstract/Free Full Text]
9. 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]
10. Sandström J, Carlsson L, Marklund SL, Edlund T. The heparin-binding domain of extracellular superoxide dismutase C and formation of variants with reduced heparin affinity. J Biol Chem. 1992; 267: 18205–18209.[Abstract/Free Full Text]
11. Karlsson K, Marklund SL. Heparin-induced release of extracellular superoxide dismutase to human blood plasma. Biochem J. 1987; 242: 55–59.[Medline] [Order article via Infotrieve]
12. Nakane H, Chu Y, Faraci FM, Oberley LW, Heistad DD. Gene transfer of extracellular superoxide dismutase increases SOD activity in cerebrospinal fluid. Stroke. 2001; 32: 184–189.[Abstract/Free Full Text]
13. Macdonald RL, Weir BKA. A review of hemoglobin and the pathogenesis of cerebral vasospasm. Stroke. 1991; 22: 971–982.[Abstract/Free Full Text]
14. Kim DE, Suh YS, Lee M-S, Kim KY, Lee JH, Lee HS, Hong KW, Kim CD. Vascular NAD (P)H oxidase triggers delayed cerebral vasospasm after subarachnoid hemorrhage in rats. Stroke. 2002; 33: 2687–2691.[Abstract/Free Full Text]
15. Chu Y, Lund DD, Faraci FM, Heistad DD. Gene transfer of extracellular superoxide dismutase reduces blood pressure of spontaneously hypertensive rats. Circulation. 2001; 104 (suppl II): 23.Abstract.
16. Chu Y, Heistad DD. Gene transfer to blood vessels using adenoviral vectors. Methods Enzymol. 2002; 346: 263–276.[CrossRef][Medline] [Order article via Infotrieve]
17. Toyoda K, Faraci FM, Russo AF, Davidson BL, Heistad DD. Gene transfer of calcitonin gene-related peptide to cerebral arteries. Am J Physiol. 2000; 278: H586–H594.
18. Spitz DR, Oberley LW. An assay for superoxide dismutase activity in mammalian tissue homogenates. Anal Biochem. 1989; 179: 8–18.[CrossRef][Medline] [Order article via Infotrieve]
19. Toyoda K, Faraci FM, Watanabe Y, Ueda T, Andresen JJ, Chu Y, Otake S, Heistad DD. Gene transfer of calcitonin gene-related peptide prevents vasoconstriction after subarachnoid hemorrhage. Circ Res. 2000; 87: 818–824.[Abstract/Free Full Text]
20. Marklund SL. Analysis of extracellular superoxide dismutase in tissue homogenates and extracellular fluids. Methods Enzymol. 1990; 186: 260–265.[Medline] [Order article via Infotrieve]
21. Gryglewski RJ, Palmer RMJ, Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature. 1986; 320: 454–456.[CrossRef][Medline] [Order article via Infotrieve]
22. Wang HD, Pagano PJ, Du Y, Cayatte AJ, Quinn MT, Brecher P, Cohen RA. Superoxide anion from the adventitia of the rat thoracic aorta inactivates nitric oxide. Circ Res. 1998; 82: 810–818.[Abstract/Free Full Text]
23. Crow JP, Beckman JS. Reaction between nitric oxide, superoxide, and peroxynitrite: footprints of peroxynitrite in vivo. Adv Pharmacol. 1995; 34: 17–43.[Medline] [Order article via Infotrieve]
24. Radi R, Beckman JS, Bush KM, Freeman BA. Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch Biochem Biophys. 1991; 288: 481–487.[CrossRef][Medline] [Order article via Infotrieve]
25. Szabó C, Zingarelli B, O’Connor M, Salzman AL. DNA strand breakage, activation of poly(ADP-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity in macrophages and smooth muscle cells exposed to peroxynitrite. Proc Natl Acad Sci U S A. 1996; 93: 1753–1758.[Abstract/Free Full Text]
26. Sakaki S, Ohta S, Nakamura H, Takeda S. Free radical reaction and biological defense mechanism in the pathogenesis of prolonged vasospasm in experimental subarachnoid hemorrhage. J Cereb Blood Flow Metab. 1988; 8: 1–8.[Medline] [Order article via Infotrieve]
27. Satoh M, Date I, Nakajima M, Takahashi K, Iseda K, Tamiya T, Ohmoto T, Ninomiya Y, Asari S. Inhibition of poly(ADP-ribose) polymerase attenuates cerebral vasospasm after subarachnoid hemorrhage in rabbits. Stroke. 2001; 32: 225–231.[Abstract/Free Full Text]
28. Katusic ZS, Schugel J, Cosentino F, Vanhoutte PM. Endothelium-dependent contractions to oxygen-derived free radicals in the canine basilar artery. Am J Physiol. 1993; 264: H859–H864.[Medline] [Order article via Infotrieve]
29. Steinke DE, Weir BK, Findlay JM, Tanabe T, Grace M, Krushelnycky BW. A trial of the 21-aminosteroid U74006F in a primate model of chronic cerebral vasospasm. Neurosurgery. 1989; 24: 179–186.[Medline] [Order article via Infotrieve]
30. Kunsch C, Medford RM. Oxidative stress as a regulator of gene expression in the vasculature. Circ Res. 1999; 85: 753–766.[Abstract/Free Full Text]
31. Laher I, Zhang JH. Protein kinase C and cerebral vasospasm. J Cereb Blood Flow Metab. 2001; 21: 887–906.[CrossRef][Medline] [Order article via Infotrieve]
32. Satoh M, Parent AD, Zhang JH. Inhibitory effect with antisense mitogen-activated protein kinase oligodeoxynucleotide against cerebral vasospasm in rats. Stroke. 2002; 33: 775–781.[Abstract/Free Full Text]
33. Ono S, Date I, Onoda K, Shiota T, Ohmoto T, Ninomiya Y, Asari S, Morishita R. Decoy administration of NF-kappaB into the subarachnoid space for cerebral angiopathy. Hum Gene Ther. 1998; 9: 1003–1011.[Medline] [Order article via Infotrieve]
34. Teixeira HD, Schumacher RI, Meneghini R. Lower intracellular hydrogen peroxide levels in cells overexpressing CuZn-superoxide dismutase. Proc Natl Acad Sci U S A. 1998; 95: 7872–7875.[Abstract/Free Full Text]
35. Handa Y, Kabuto M, Kobayashi H, Kawano H, Takeuchi H, Hayashi M. The correlation between immunological reaction in the arterial wall and the time course of the development of cerebral vasospasm in a primate model. Neurosurgery. 1991; 28: 542–549.[CrossRef][Medline] [Order article via Infotrieve]
36. Megyesi JF, Vo llrath B, Cook DA, Findlay JM. In vivo animal models of cerebral vasospasm: a review. Neurosurgery. 2000; 46: 448–461.[CrossRef][Medline] [Order article via Infotrieve]
37. Young B, Runge JW, Waxman KS, Harrington T, Wilberger J, Muizelaar JP, Boddy A, Kupiec JW. Effects of pegorgotein on neurologic outcome of patients with severe head injury: a multicenter, randomized controlled trial. JAMA. 1996; 276: 538–543.[Abstract]
38. Gunnet CA, Heistad DD. Virally mediated gene transfer to the vasculature. Microcirculation. 2002; 9: 23–34.[CrossRef][Medline] [Order article via Infotrieve]

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