Rosuvastatin Ameliorates Early Brain Injury after Subarachnoid Hemorrhage via Suppression of Superoxide Formation and Nuclear Factor-Kappa B Activation in Rats Ken Uekawa, MD,* Yu Hasegawa, MD, PhD,* Mingjie Ma, MD,* Takashi Nakagawa, MD,* Tetsuji Katayama, MD,* Daisuke Sueta, MD,* Kensuke Toyama, MD, PhD,* Keiichiro Kataoka, MD, PhD,* Nobutaka Koibuchi, PhD,* Takayuki Kawano, MD, PhD,† Jun-ichi Kuratsu, MD, PhD,† and Shokei Kim-Mitsuyama, MD, PhD*

Background: Statins, or 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, have been suggested to possess pleiotropic effects, including antioxidant and anti-inflammatory properties. We investigated the protective effects of pretreatment with rosuvastatin, a relatively hydrophilic statin, on early brain injury (EBI) after a subarachnoid hemorrhage (SAH), using the endovascular perforation SAH model. Methods: Eighty-six male Sprague–Dawley rats were randomly divided into 3 groups: (1) sham operation, (2) SAH 1 vehicle, and (3) SAH 1 10 mg/kg rosuvastatin. Rosuvastatin or vehicle was orally administered to rats once daily from 7 days before to 1 day after the SAH operation. After SAH, we examined the effects of rosuvastatin on the neurologic score, brain water content, neuronal cell death estimated by terminal deoxynucleotidyl transferase–mediated uridine 50 -triphosphate nick end labeling staining, blood–brain barrier disruption by immunoglobulin G (IgG) extravasation, oxidative stress, and proinflammatory molecules. Results: Compared with the vehicle group, rosuvastatin significantly improved the neurologic score and reduced the brain water content, neuronal cell death, and IgG extravasation. Rosuvastatin inhibited brain superoxide production, nuclear factor-kappa B (NF-kB) activation, and the increase in activated microglial cells after SAH. The increased expressions of tumor necrosis factor-alpha, endothelial matrix metalloproteinase-9, and neuronal cyclooxygenase-2 induced by SAH were prevented by rosuvastatin pretreatment. Conclusions: The present study demonstrates that rosuvastatin pretreatment ameliorates EBI after SAH through the attenuation of oxidative stress and NF-kB–mediated inflammation. Key Words: Subarachnoid hemorrhage—early brain injury—rosuvastatin—reactive oxygen species—nuclear factor-kappa B. Ó 2013 by National Stroke Association

From the *Department of Pharmacology and Molecular Therapeutics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto; and †Department of Neurosurgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan. Received September 14, 2013; revision received October 29, 2013; accepted December 3, 2013. Grant information: Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (23390058). This work was also supported in part by a research grant from AstraZeneca.

The authors declare no competing financial interests. Address correspondence to Shokei Kim-Mitsuyama, MD, PhD, FAHA, Department of Pharmacology and Molecular Therapeutics, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan. E-mail: kimmitsu@ gpo.kumamoto-u.ac.jp. 1052-3057/$ - see front matter Ó 2013 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2013.12.004

Journal of Stroke and Cerebrovascular Diseases, Vol. -, No. - (---), 2013: pp 1-11

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Introduction Aneurysmal subarachnoid hemorrhage (SAH) is a devastating neurologic condition with poor outcomes and high mortality. Although previous studies on the pathogenesis of SAH have focused mainly on the delayed cerebral vasospasm, the functional outcome is not improved even if the angiographic vasospasm is reversed.1 Importantly, early brain injury (EBI), which occurs within 72 hours of cerebral aneurysm rupture, has been reported to be involved in the poor outcomes after SAH more often than cerebral vasospasm.2 Regarding the pathogenesis of EBI post-SAH, an aneurysmal rupture induces the mechanical compression of brain tissue, accumulation of subarachnoid blood, and global cerebral ischemia associated with an increased intracranial pressure, and these changes result in oxidative stress, inflammation, and blood–brain barrier (BBB) disruption, which finally lead to brain edema and neuronal cell death.2 Statins, inhibitors of the 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase, are widely used as cholesterol-lowering drugs and have been suggested to have pleiotropic effects, including antioxidative stress and anti-inflammatory properties.3 The molecular mechanism comes from the inhibition of geranylgeranylation of the small G-proteins.3 However, the full effects of the statins on EBI after SAH remain unknown. Rosuvastatin is known as a potent statin and strongly inhibits HMGCoA reductase activity.4 Moreover, rosuvastatin is reported to possess neuroprotective effects in experimental stroke models, including the focal cerebral ischemia model5-7 and the stroke-prone hypertensive rat.8 In addition, an in vitro study showed that rosuvastatin protected cortical neurons from N-methyl-D-aspartate–induced excitotoxicity.9 However, limited research is available that examines the effects of rosuvastatin in the context of EBI and SAH. Therefore, the present work set out to demonstrate that rosuvastatin can attenuate EBI in experimentally induced SAH via antioxidative stress and anti-inflammatory processes.

Materials and Methods Animals and Drugs All animal experiments were approved by the Kumamoto University Committee for Laboratory Animal Care and Use. Male Sprague–Dawley rats (Charles River, Yokohama, Japan) weighing 380-430 g were used for this study. Eighty-six rats were randomly divided into 3 groups: (1) sham operation group (n 5 19), (2) SAH 1 vehicle (n 5 35), and (3) SAH 1 10 mg/kg rosuvastatin (n 5 32). Because 10 mg/kg dosage of rosuvastatin is required for approximately 90% inhibition of HMG-CoA reductase activity in rodents,4,7 we chose the 10 mg/kg dosage of rosuvastatin. Rosuvastatin was

orally administered by gastric gavage once daily for 7 days before and at 22 hours after SAH operation to maintain drug levels. We assessed the neurologic deficits at 24 hours after SAH operation and euthanized immediately after the neurologic evaluation. Rosuvastatin was kindly provided by AstraZeneca (Macclesfield, London, UK).

Induction of SAH Rats were anesthetized with 1.5%-2% isoflurane through a face mask. The rectal temperature was maintained at 37.0 6 .5 C by a feedback-regulated heating system during surgery. The endovascular perforation model of SAH was used as previously described10 with some modifications. Briefly, the left common carotid artery was exposed, and the left external carotid artery (ECA) was identified. The ECAwas dissected to 4-5 mm distally from the common carotid artery bifurcation and reflected caudally. The ECA origin was temporarily occluded with an aneurysm clip while ensuring patency of the internal carotid artery (ICA). A hollow polytetrafluoroethylene tube (SUBL-120; Braintree Scientific, Braintree, MA) was inserted into the left ECA and advanced until resistance was felt through the left ICA to the intracranial ICA bifurcation. A tungsten rod (diameter, .004 inch; A-M systems, Sequim, WA) was advanced through the microtube 3 mm farther than the tip of the microtube to perforate the artery at the ICA bifurcation. Then, the microtube and rod were withdrawn through the ECA. The sham-operated rats underwent an identical procedure except for the arterial puncture.

Physiological Parameters A catheter was inserted in the femoral artery and used for the continuous recording of arterial pressure and blood sampling. The pH levels and the arterial pO2 and pCO2 were measured using a blood gas analyzer (IRMA TRUpoint analyzer; International Technidyne Corporation, Edison, NJ) before and 3 and 30 minutes after SAH induction. The animals used in the evaluation of the physiological parameters were not used for further study because of the influence of measuring the physiological parameters, such as insertion of the catheter, loss of blood, etc.

Neurologic Scoring Using a 22-point scoring system with modifications of the method described by Garcia et al,11 we assessed neurologic deficits in a blinded manner of each animal before euthanasia as previously described.12 This evaluation consisted of 7 tests: spontaneous activity, symmetrical movements of limbs, forelimbs outstretching, wall climbing of a wire cage, axillary touch response, vibrissae touch response, and beam walking. A higher score indicated greater neurologic function.

ROSUVASTATIN INHIBITS BRAIN INJURY AFTER SAH

SAH Grade We evaluated the SAH severity in a blinded manner with an 18-point SAH grading system at the time of euthanasia as previously described.12,13 Briefly, the subarachnoid cistern was divided into 6 parts in photographs of the base of the brain, and each part was subscored according to the presence of blood in the subarachnoid space from 0 to 3 (total score: 0-18)—grade 0: no subarachnoid blood; grade 1: minimal subarachnoid blood; grade 2: moderate blood clot with recognizable arteries; and grade 3: blood clot obliterating all arteries.

Brain Water Content The rats were decapitated at 24 hours after SAH induction, and the brain water content was evaluated as previously described.12,14 Briefly, the brains were quickly removed and divided into right and left cerebral hemispheres, cerebellum, and brain stem. The weight of each brain sample was immediately measured (wet weight). The brain samples were then heated in an oven at 80 C for 72 hours, and the sample weights were measured again (dry weight). The following formula was used to calculate the percentage of brain water content: [(wet weight 2 dry weight)/wet weight] 3 100 (%).

Western Blot Our detailed Western blot method has been previously described.15 Briefly, brain protein extracts (20 mg) from the left hemisphere were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were then probed with the following primary antibodies: antiphosphorylated-Ser536 p65 (1:5000; Cell Signaling Technology, Danvers, MA), anti-p65 (1:5000; Cell Signaling Technology), antitumor necrosis factor-alpha (TNF-a, 1:2500; Cell Signaling Technology), anti-metalloproteinase9 (MMP-9, 1:5000; Abcam, Cambridge, MA), anti-cyclooxygenase-2 (COX-2, 1:5000; Acris, Herfod, Germany), and anti-a-tubulin (1:5000; Calbiochem, Billerica, MA). The membranes were incubated for 1 hour with appropriate secondary antibodies. Protein bands were visualized using an enhanced chemiluminescence reagent kit (Amersham ECL plus kit, GE Healthcare, UK), and protein quantification was performed by optical density methods using ImageJ version 1.46 software (National Institutes of Health, Bethesda, MD). For each sample, the protein expression was normalized to the expression of a-tubulin and compared with the mean value of the sham group.

Immunohistochemistry Immunohistochemistry was performed on 8-mm frozen coronal brain sections as previously described.16 Briefly, sections at 2.5 mm posterior to bregma were fixed with 4% paraformaldehyde, incubated with blocking solution

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for 30 minutes, and incubated overnight at 4 C with the following primary antibodies: anti-p65 (1:500; Cell Signaling Technology), anti-neuronal nuclei (NeuN, 1:1000; Chemicon, Temecula, CA), anti-platelet endothelial cell adhesion molecule-1 (PECAM-1, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-glial fibrillary acidic protein (1:200; Santa Cruz Biotechnology), anti-ionized calcium-binding adaptor molecule-1 (Iba-1, 1:200; Abcam), anti-MMP-9 (1:200; Abcam), and anti-COX-2 (1:300; Acris). The reaction product was visualized with 3,30 -diaminobenzidine or Alexa Fluor 488 (green) and 594 (red) conjugated secondary antibodies (1:200; Molecular Probes, Eugene, OR). The numbers of Iba-1–positive cells and COX-2–positive cells in each tissue sample were counted in 3 fields of view of the left cortex in a blinded manner and expressed as the mean of the positive cells per square millimeter.

Measurement of Immunoglobulin G Extravasation Brain immunolocalization of immunoglobulin G (IgG) was evaluated as previously described.17,18 Briefly, the brain sections were incubated in the presence of .3% H2O2 and with rabbit anti-rat IgG–horseradish peroxidase (Invitrogen, Carlsbad, CA) for 1 hour. After washing with PBS, the reaction product was visualized with 3,30 diaminobenzidine. IgG immunoreactivity was quantified in 3 fields of view of the left cortex with the Lumina Vision version 2.2.0 analysis software (Mitani Corporation, Tokyo, Japan) in a blinded manner and expressed as the mean value compared with the sham group.

Detection of Superoxide Levels Dihydroethidium (DHE; Sigma, St Louis, MO) was used to evaluate brain superoxide levels in situ as previously described.19 Briefly, sections without paraformaldehyde fixation were incubated with DHE (5 mmol/L) for 30 minutes at 37 C in the dark. DHE fluorescence was visualized using fluorescent microscopy (FluoView FV1000; Olympus, Tokyo, Japan) at an excitation wavelength of 559 nm. The detector and laser settings were kept constant for all samples analyzed. The DHE fluorescence was quantified in 3 fields of view of the left cortex with the Lumina Vision software in a blinded manner and expressed as the mean value compared with the sham group.

Terminal Deoxynucleotidyl Transferase–Mediated Uridine 50 -Triphosphate Nick End Labeling The brain sections were immunostained with antiNeuN antibody (1:200; Chemicon) and then were subjected to terminal deoxynucleotidyl transferase– mediated uridine 50 -triphosphate nick end labeling (TUNEL) staining with an in situ cell death detection kit (Roche Inc., Mannheim, Germany) as previously described.12 Then, the sections were mounted with

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4 -6-diamidino-2-phenylindole. The incubation with labeling solution without the enzyme served as a negative control. The number of left cortical TUNELpositive neurons was counted in 3 fields of view in a blinded manner and expressed as the mean number of TUNEL-positive neurons per square millimeter.

Statistical Analysis The SAH grade and neurologic scores were expressed as the median and interquartile range and were analyzed using the Kruskal–Wallis test followed by Steel–Dwass post hoc test for multiple comparisons. The mortality rate was analyzed with the chi-square test. Other values were expressed as the mean 6 SD. Normality was tested with the Kolmogorov–Smirnov test, and Bartlett test was performed to examine whether variances were similar across comparison groups. When data were normally distributed and variances were similar across comparison groups, the statistical significance of differences was assessed with a 1-way analysis of variance followed by the Newman–Keuls post hoc test for multiple comparisons. When a normal distribution was not confirmed or similar variances were not obtained among comparison groups, data were analyzed with the Kruskal–Wallis test followed by Steel–Dwass post hoc test for multiple comparisons. Physiological data acquired during the SAH operation were analyzed using a 2-way analysis of variance with repeated measures followed by Bonferroni post hoc test for multiple comparisons. P less than .05 was considered significant. The method of statistical analysis used in each experiment is described in all figure legends. The statistical analyses were performed with GraphPad Prism version 5.02 for Windows (GraphPad Software Inc., San Diego, CA) and Ekuseru-Tokei 2012

statistical software (Social Survey Research Information Co, Ltd, Tokyo, Japan).

Results Physiological Data Physiological parameters were monitored in the sham, vehicle, and rosuvastatin groups (n 5 5 per group). Although the SAH animals showed significantly lower pH levels than the sham group at 3 minutes after SAH induction, no significant differences were observed between the vehicle and the rosuvastatin groups for the mean arterial blood pressure, heart rate, and arterial blood gases (pH levels, pO2, and pCO2) before and 3 and 30 minutes after SAH induction (Table 1).

Mortality and SAH Grade The 24-hour mortality rate after SAH was not significantly different between the vehicle (30.0% [9 of 30 rats]) and rosuvastatin (25.9% [7 of 27 rats]) groups. No sham-operated rats died. Considering that the brain injury of the endovascular perforation model is variable and is related to the bleeding volume,13 the SAH rats were divided into 2 categories according to the SAH grade: mild (SAH grades from 0 to 7) and severe (SAH grades from 8 to 18). As in previous studies,12,14,20 our study showed that the brain edema and neurobehavioral deficits in the mild group were very similar to that of the sham group (data not shown). Thus, 13 mild SAH rats (SAH grades: 0-7) were then excluded from this study (vehicle 7 rats; rosuvastatin 6 rats), and the severe SAH rats (SAH grades: 8-18) were included in the following studies (n 5 14 per group).

Table 1. Physiological parameters measured during SAH operation

Sham Pre-SAH 3 min post-SAH 30 min post-SAH Vehicle Pre-SAH 3 min post-SAH 30 min post-SAH Rosuvastatin (10 mg/kg) Pre-SAH 3 min post-SAH 30 min post-SAH

Mean BP (mmHg)

HR (per min)

pH

pO2 (mmHg)

pCO2 (mmHg)

87.6 6 7.4 89.9 6 9.6 87.3 6 4.9

340.8 6 50.5 380.4 6 26.4 363.6 6 39.7

7.44 6 .03 7.41 6 .04 7.39 6 .03

161.3 6 21.7 151.4 6 17.6 161.4 6 13.9

41.7 6 3.2 44.9 6 5.4 48.3 6 5.6

86.9 6 4.3 102.7 6 15.0 77.0 6 12.4

351.6 6 40.6 344.4 6 47.2 346.8 6 62.3

7.43 6 .03 7.34 6 .06* 7.34 6 .05

166.9 6 20.1 131.2 6 55.3 159.0 6 16.2

44.1 6 3.5 53.6 6 9.1 53.0 6 6.6

85.1 6 6.4 101.0 6 18.8 75.5 6 9.9

382.8 6 57.3 340.8 6 52.2 355.2 6 36.8

7.43 6 .05 7.33 6 .06* 7.36 6 .04

173.1 6 27.6 156.9 6 26.0 168.2 6 23.9

45.2 6 5.5 54.6 6 11.9 52.4 6 8.5

Abbreviations: BP, blood pressure; HR, heart rate; SAH, subarachnoid hemorrhage. Values are presented as the mean 6 SD (n 5 5 in each group). Statistical analysis was performed with a 2-way analysis of variance with repeated measures followed by Bonferroni post hoc test for multiple comparisons. *P , .05 versus sham at 3 minutes post-SAH.

ROSUVASTATIN INHIBITS BRAIN INJURY AFTER SAH

The SAH grading scores were similar between the vehicle and rosuvastatin groups (Fig 1, A).

The Effects of Rosuvastatin on Neurologic Score and Brain Water Content We evaluated the neurologic scores at 1 hour before operation, and the neurologic scores were quite similar between the groups (data not shown). At 24 hours after SAH compared with the sham group, the neurologic scores had deteriorated significantly in the vehicle group (22 [21-22] versus 14 [10-15.8]; P , .01, Fig 1, B). Rosuvastatin significantly reduced the neurologic deficits (18.5 [16.5-19.3]; P , .05) at 24 hours after SAH compared with the vehicle group. As shown in Figure 1, C, the left hemisphere brain water content in the vehicle group was significantly increased compared with the sham group at 24 hours after SAH (79.1 6 .4% versus 78.3 6 .2%; P , .01). Rosuvastatin significantly reduced the increase in brain water content induced by SAH (78.7 6 .3%; P , .05) compared with the vehicle group.

The Effects of Rosuvastatin on Neuronal Cell Death and BBB Disruption As shown in Figure 2, A, a large number of TUNELpositive neurons were observed in the left cortex of the vehicle group at 24 hours after SAH. The number of TUNEL-positive neurons was reduced in the rosuvastatin group compared with the vehicle group (253.2 6 76.6/ mm2 versus 423.3 6 99.3/mm2; P , .05). As presented in Figure 2, B, compared with the sham group, the density of IgG staining was greater in the vehicle group at 24 hours after SAH (P , .01). In the rosuvastatin group, the density of IgG staining was reduced compared with the vehicle group (P , .05).

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The Effects of Rosuvastatin on Superoxide Levels and Nuclear Factor-Kappa B Activation As evaluated by DHE staining, the superoxide levels were increased in the left cortex of the vehicle group compared with the sham group at 24 hours after SAH (Fig 3, A, P , .01), and rosuvastatin significantly attenuated these levels compared with the vehicle group (P , .05). To examine nuclear factor-kappa B (NF-kB) activation in the brain after SAH, we evaluated the nuclear translocation of p65, a NF-kB subunit. Double immunohistochemical staining revealed that p65 translocated into the nucleus in Iba-1–, PECAM-1–, and NeuN-positive cells after SAH (Fig 3, B). Western blot analysis revealed that p65 phosphorylation, an indicator of NF-kB activation, was enhanced in the vehicle group compared with the sham group at 24 hours after SAH (Fig 3, C, P , .01). However, rosuvastatin significantly suppressed p65 phosphorylation compared with the vehicle group (P , .05), whereas the expression of total p65 was comparable between all groups.

The Effects of Rosuvastatin on Microglial Activation and TNF-a Expression As shown in Figure 4, A, the number of Iba-1–positive cells was significantly increased in the vehicle group compared with the sham group at 24 hours after SAH (P , .01), and rosuvastatin treatment significantly reduced the Iba-1–positive cells in the left cortex at 24 hours after SAH (P , .01). Western blot analysis demonstrated that TNF-a expression was significantly upregulated in the vehicle group compared with the sham group at 24 hours after SAH (Fig 4, B, P , .01), and rosuvastatin significantly decreased this TNF-a upregulation compared with the vehicle group (P , .01).

Sham Vehicle RSV

NS

10 5 0

Sham Vehicle

RSV

20 15 10 5 0

P

Rosuvastatin ameliorates early brain injury after subarachnoid hemorrhage via suppression of superoxide formation and nuclear factor-kappa B activation in rats.

Statins, or 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, have been suggested to possess pleiotropic effects, including antioxidant and ...
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