nitrative injury to cerebral microvessels after global cerebral ischemia

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Effects of crocin on reperfusion-induced oxidative/nitrative injury to cerebral microvessels after global cerebral ischemia Yong-Qiu Zheng⁎, Jian-Xun Liu, Jan-Nong Wang, Li Xu Research Center, Xiyuan Hospital, China Academy of Chinese Medical Sciences, 1, Xi Yuan yard Road, Haidian District, Beijing 100091, PR China

A R T I C LE I N FO

AB S T R A C T

Article history:

This paper studied the effects of crocin, a pharmacologically active component of Crocus

Accepted 20 December 2006

sativus L., on ischemia/reperfusion (I/R) injury in mice cerebral microvessels. Transient

Available online 29 December 2006

global cerebral ischemia (20 min), followed by 24 h of reperfusion, significantly promoted the generation of nitric oxide (NO) and malondialdehyde (MDA) in cortical microvascular

Keywords:

homogenates, as well as markedly reduced the activities of superoxide dismutase (SOD) and

Crocin

glutathione peroxidase (GSH-px) and promoted the activity of nitric oxide synthase (NOs).

G protein-coupled receptor kinase

Reperfusion for 24 h led to serous edema with substantial microvilli loss, vacuolation,

Nitric oxide synthase

membrane damage and mitochondrial injuries in cortical microvascular endothelial cells

Superoxide dismutase

(CMEC). Furthermore, enhanced phosphorylation of extracellular signal-regulated kinase 1/

Glutathione peroxidase

2 (ERK1/2) and decreased expression of matrix metalloproteinase-9 (MMP-9) were detected in cortical microvessels after I (20 min)/R (24 h). Reperfusion for 24 h also induced membrane (functional) G protein-coupled receptor kinase 2 (GRK2) expression, while it reduced cytosol GRK2 expression. Pretreatment with crocin markedly inhibited oxidizing reactions and modulated the ultrastructure of CMEC in mice with 20 min of bilateral common carotid artery occlusion (BCCAO) followed by 24 h of reperfusion in vivo. Furthermore, crocin inhibited GRK2 translocation from the cytosol to the membrane and reduced ERK1/2 phosphorylation and MMP-9 expression in cortical microvessels. We propose that crocin protects the brain against excessive oxidative stress and constitutes a potential therapeutic candidate in transient global cerebral ischemia. © 2006 Elsevier B.V. All rights reserved.

1.

Introduction

Transient global cerebral ischemia which can occur during asphyxiation, shock, brain injury and cardiac arrest is a devastating event that is associated with great morbidity (Madl and Holzer, 2004). In certain clinical situations transient global cerebral ischemia can either be anticipated or is even induced iatrogenically such as during cardiac or thoracic surgery. Furthermore, intraoperative hypothermia during surgery with its proven neuroprotective effect is employed less frequently nowadays so that any pharmacologic neuro-

protection aimed at preventing ischemic reperfusion injuries in transient global cerebral ischemia should be the only alternative (Weigl et al., 2005). There are cumulative evidence suggesting the involvement of reoxygenation during experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents (Chan, 2001). Intense superoxide, nitric oxide (NO), and peroxynitrite formation on microvessels and surrounding end-feet may lead to cerebral hemorrhage and edema by disrupting microvascular integrity and breakdown of the blood–brain barrier (BBB). Such agents preventing ischemic

⁎ Corresponding author. Fax: +86 10 6288 6691. E-mail address: [email protected] (Y.-Q. Zheng). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.12.064

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reperfusion injuries in cerebral microvessels and diminishing oxidative/nitrative stress may reduce reperfusion-induced injury and may extend the therapeutic targets (Fagan et al., 2004; Gursoy-Ozdemir et al., 2004; Maier et al., 2006). Crocin, a pharmacologically active component of Crocus sativus L., may be a pharmacological agent that, when administered before common clinical situations, results in transient global cerebral ischemia because of its antioxidant effects (Lee et al., 2005). C. sativus L. is commonly known as saffron and it is used in traditional Chinese medicine as an antispasmodic, anticatarrhal, nerve sedative, stimulant, etc. Modern pharmacological studies have demonstrated that saffron extract or its active constituents have antitumour effects, radical scavenger properties, hypolipaemic effects and memory-improved effects (He et al., 2005; Sugiura et al., 1994; Zhang et al., 1994). It was recently reported that pretreatment of saffron extract 7 days before middle cerebral artery occlusion (MCAO) modulated cerebral malondialdehyde (MDA), glutathione peroxidase, superoxide dismutase (SOD), catalase (CAT), and Na(+),K(+)-ATPase activities, and glutamate (Glu) and aspartate (Asp) (Saleem et al., 2006). Among the constituents of saffron, crocin is the most abundant and with established antioxidant effects (Abdullaev, 1993). Further study indicated that crocin is an antioxidant, more potent than α-tocopherol, against oxidative stress-induced death of neurons and that it inhibited the formation of peroxidized lipids and partly restored SOD activity (Ochiai et al., 2004). Further investigations on molecular targets of crocin may give new insight into the mechanism underlying prophylaxis transient global cerebral ischemia. Two animal models, i.e. (1) global or bilateral common carotid artery occlusion (BCCAO) and (2) focal or MCAO, have been extensively used in studying the impact of ischemic brain injury. The BCCAO model simulates the effects produced by the systemic decrease in blood flow, affects the entire brain, and resembles transient global cerebral ischemia in clinical in a number of pathological aspects (Huang and McNamara, 2004; Shah et al., 2005). Therefore, the specific aim of this study was to evaluate the beneficial actions of crocin in ischemia/reperfusion-induced brain damage using BCCAO. In the present study, we demonstrate that crocin retrieved reperfusion-induced oxidative/nitrative injuries to cerebral microvessels after global cerebral ischemia. Further, the present study is the first, to our knowledge, to examine G protein-coupled receptor kinase 2 (GRK2) subcellular distribution in cerebral microvessels after global cerebral ischemia– reperfusion and to evaluate the effects of crocin on GRK2 subcellular distribution.

2.

87

Crocin (20, 10 mg/kg) pretreatment resulted in a significant decrease in MDA content (General F = 36.232; General P = 0.000) and a partial elevation in total antioxidant capacity including SOD activities (General F = 8.578; General P = 0.000) and GSH-px activities (General F = 5.346; General P = 0.001). Results from regression–linear test indicated that the effects of crocin on MDA contents (R = 0.480; P < 0.01) and SOD activities (R = 0.551; P < 0.01) showed dose-dependent relation, whereas the effects of crocin on GSH-px activities did not present dose-dependent relation (R = 0.191; P > 0.05) (Figs. 1 and 2).

2.2. Effect of crocin on NO content and nitric oxide synthase (NOs) activities in cortical microvascular homogenates NO content and NOs activities in cortical microvascular homogenates were significantly increased in transient global ischemia mice. Oral administration of crocin (20, 10 mg/kg) significantly inhibited the increased NO (General F = 13.007; General P = 0.000) content and NOs activities (General F = 15.794; General P = 0.000). Results from regression–linear test indicated that the effects of crocin on NO contents showed a dose-dependent relation (R = 0.429; P < 0.05), whereas the effects of crocin on NOs activities did not present a dosedependent relation (R = 0.335; P > 0.05) (Figs. 3 and 4).

2.3. Effects of crocin on the ultrastructural morphology of cortical microvascular endothelial cells (CMEC) Cortical microvessels in sham rats have capillary integrity with normal cerebromicrovascular endothelial cells, basal lamina and astrocyte end-feet. CMEC in ischemia/reperfusion (I/R) mice appeared to be serous edema with substantial

Results

2.1. Effect of crocin on MDA content and SOD, glutathione peroxidase (GSH-px) activities in cortical microvascular homogenates Cortical microvascular peroxidation, measured as MDA, was significantly increased after 20 min of BCCAO with 24 h of reperfusion, while SOD and GSH-px activities decreased.

Fig. 1 – Effects of crocin on MDA levels in cerebral microvascular homogenates after 20 min of BCCAO with 24 h of reperfusion. Statistical analysis was performed using one-way ANOVA followed by Games–Howell test for multiple comparisons. Values are mean ± SD. Each group consists of 10 rats. ***p < 0.001, compared with I/R group; ###p < 0.001, compared with I/R group treated with crocin (5 mg/kg). General F = 36.232; General P = 0.000.

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Fig. 2 – Effects of crocin on SOD and GSH-px activities in cerebral microvascular homogenates after 20 min of BCCAO with 24 h of reperfusion. Statistical analysis was performed using one-way ANOVA followed by Tukey's test for multiple comparisons. Values are mean ± SD. Each group consists of 10 rats. *p < 0.05, **p < 0.01, ***p < 0.001, compared with I/R group; #p < 0.05 compared with I/R group treated with crocin (5 mg/kg). General F in GSH-px test = 5.346; General P in GSH-px test = 0.001; General F in SOD test = 8.578; General P in SOD test = 0.000.

microvilli loss. Capillary integrity was destroyed, including perivascular edema and vacuolation and membrane damage. Intracellularly, the nuclear of CMEC was swollen. The mitochondria decreased and swelled with the ridges decreasing. Rough endoplasmic reticulum (RER) decreased and dilated with degranulation. Oral administration of crocin (20, 10 mg/kg) repaired the injury mentioned above (Fig. 5).

Fig. 4 – Effects of crocin on NOs activities in cerebral microvascular homogenates after 20 min of BCCAO with 24 h of reperfusion. Statistical analysis was performed using one-way ANOVA followed by Games–Howell test for multiple comparisons. Values are mean ± SD. Each group consists of 10 rats. ***p < 0.001 compared with I/R group; #p < 0.05 compared with I/R group treated with crocin (5 mg/kg). General F = 15.794; General P = 0.000.

2.4. Effects of crocin on extracellular signal-regulated kinase (ERK) phosphorylation and matrix metalloproteinase-9 (MMP-9) expression in cortical microvessels ERK1/2 phosphorylation and MMP-9 expression in cortical microvascular homogenates were determined by Western blot analysis. It was found that I/R induced more phosphorylation of ERK1/2 and more expression of MMP-9 than those in sham controls. Crocin (20, 10, 5 mg/kg) pretreatment in vivo inhibits ERK1/2 phosphorylation and MMP-9 expression. The expression of total ERK was not changed (Fig. 6).

2.5. Effects of crocin on GRK2 subcellular distribution in cortical microvessels To examine the roles of GRK2 in brain microvascular damage after transient cerebral ischemia, we detect subcellular distribution of GRK2. The GRK2 levels were significantly decreased in the soluble fraction (cytosol) and increased in the particulate fraction (membrane) after global cerebral ischemia–reperfusion. Crocin (20, 10 mg/kg) pretreatment in vivo not only reduced membrane (functional) GRK2 expression but also increased cytosol GRK2 expression, suggesting its ability to inhibit GRK2 translocation from the cytosol to the membrane (Fig. 7).

Fig. 3 – Effects of crocin on NO levels in cerebral microvascular homogenates after 20 min of BCCAO with 24 h of reperfusion. Statistical analysis was performed using one-way ANOVA followed by Tukey's test for multiple comparisons. Values are mean ± SD. Each group consists of 10 rats. *p < 0.05, **p < 0.01, ***p < 0.001, compared with I/R group; General F = 13.007; General P = 0.000.

3.

Discussion

To our knowledge, this is the first report that provided evidence of antioxidant effects of crocin on brain microvessels in a mice transient cerebral ischemia model. In the present study, we found that crocin markedly inhibited oxidizing reactions and modulated the ultrastructure of CMEC in mice

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89

Fig. 5 – Effects of crocin on the ultrastructural morphology of CMEC. Rats were sacrificed on day 21. Rat cortical capillary fragments were fixed with 2.5% glutaraldehyde solution overnight and fixing with 1% osmic acid for 2 h. Being embedded in an Epon/Araldite mixture and stained with uranyl acetate and lead citrate, the cerebral microvessels were observed under a 1230 type transmission electron microscope. (A) Cerebral microvessels in sham rats with normal microvascular endothelial cells (a), basal lamina (b) and astrocyte end-feet (c). (B) CMEC was serous edema after 20 min of BCCAO with 24 h of reperfusion. The mitochondria decreased and swelled with ridges decreasing (a). Rough endoplasmic reticulum (RER) decreased and dilated with degranulation (b). (C) In I/R rats treated with crocin (20 mg/kg), the swelling of CMEC was decreased (a). The destroyed capillary integrity was restored (b). (D) In I/R rats treated with crocin (10 mg/kg), the swelling of CMEC decreased.

with 20 min of BCCAO followed by 24 h of reperfusion. Furthermore, we showed that crocin modulated GRK2 subcellular distribution and inhibited ERK1/2 phosphorylation and MMP-9 expression in cortical microvessels. The results from the present study support the protective effects of crocin in transient global cerebral ischemia. BCCAO ischemia in the rat produces distinct patterns of BBB opening depending on ischemic duration (Preston and Webster, 2004). 20 min of prolonged ischemia may cause a dramatic deterioration of BBB in cerebral cortex between the 6and 24-h time points (Preston and Webster, 2002) and much greater edema. Preston et al. demonstrated a moderate BBB leakiness and mitochondrial injuries in cortex of rats after 20 min of BCCAO with 24 h of reperfusion (Preston et al., 1993). In the present study, we chose to measure ultrastructural morphology of CMEC in cortex of mice after 20 min of BCCAO with 24 h of reperfusion. The injury in our global model appeared to be serous edema with the substantial microvilli loss, vacuolation, membrane damage and mitochondrial injury. Pretreatment of crocin at the doses of 20 and 10 mg/ kg repaired the injury mentioned above. The results suggest that crocin has protective effects to moderate BBB leakiness in the ischemic brain of C57BL/6J mice. Recent studies have confirmed the pivotal role of oxidative stress in the pathogenesis of acute ischemic stroke (del Zoppo, 2006; Ryter and Choi, 2005; Schaller, 2005). Transient cerebral ischemia (30 min), followed by 1–24 h of reperfusion,

significantly increased the generation of reactive oxygen species (ROS), NO, and lipid peroxidation end-products, as well as markedly reduced the levels of the endogenous antioxidant glutathione (Collino et al., 2006). Furthermore, ROS can activate diverse downstream signaling pathways, such as mitogen-activated protein kinases (MAPKs) and the transcription factor nuclear factor-kappa B (NF-κB), thus regulating the expression of a variety of proinflammatory proteins (Crack and Taylor, 2005). Supporting this view, scavengers of ROS or inhibitors of lipid peroxidation protect the brain after ischemia–reperfusion (Chan, 2001; Kontos, 2001). ROS can be scavenged by endogenous antioxidants, involving the cooperative action of SOD, CAT and GSH-px (Kakita et al., 2002; White et al., 2000). Mice overexpressing SOD are more resistant to ischemia associated with reperfusion, and conversely, larger infarcts develop in SOD knockout mice (Kim et al., 2002; Kondo et al., 1997; Yang et al., 1994). In an earlier report, saffron exerted its function of inhibiting lipid peroxidation by increasing SOD, CAT and GSH-px (Premkumar et al., 2003). Further studies also reported that safranal, an active constituent of saffron, may ameliorate I/R injuryinduced oxidative damage in rat hippocampus and reversed the increase of MDA levels (Hosseinzadeh and Sadeghnia, 2005). In the present study, we assessed the effects of crocin on lipid peroxidation, which was measured in terms of SOD, GSHpx and MDA. The SOD and GSH-px activities decreased and MDA levels increased significantly following transient global

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Fig. 6 – Effects of crocin on ERK phosphorylation and MMP-9 expression in cerebral microvessels. Upper panel: Immunoblots of phosphorylated ERK1/2 and MMP-9 expression in cortical microvascular homogenates which were determined as described in the text. Lower panel: Bar graphs show quantitative evaluation of ERK1/2 phosphorylation and MMP-9 expression. I/R caused an increase in the ERK1/2 phosphorylation and MMP-9 expression. Crocin treatment led to the decrease in the amount of ERK1/2 phosphorylation and MMP-9 expression. Results were obtained from three independent experiments; only one representative experiment is shown, n = 3. Data are mean ± SD. Nonparametric test was used for statistical analysis. *p < 0.05 compared with I/R group. Lane 1: sham; lane 2: I/R model; lane 3: I/R + crocin (20 mg/kg); lane 4: I/R + crocin (10 mg/kg); lane 5: I/R + crocin (5 mg/kg).

cerebral ischemia. Crocin reversed the increase of MDA levels and the decrease of SOD and GSH-px activities, thereby confirming its antioxidant role in brain I/R. Furthermore, SOD normally prevents decomposition of released NO. During reperfusion, NO mainly produced by eNOs can diffuse and react with superoxide generated in end-feet and endothelia to form peroxynitrite around microvessels (Gursoy-Ozdemir et al., 2000). On the other hand, the major sources of NO were nNOs expressing in neurons and endothelia in the mouse brain 3 h after reperfusion (del Zoppo et al., 1998; Gursoy-Ozdemir et al., 2004; Yoshida et al., 1995). Excessive NO generation by nNOs is cytotoxic and nNOs knockouts are resistant to ischemia (Huang et al., 1994). It was reported that hypoxia-induced BBB breakdown can be diminished by a decrease in NO and that inhibition of NO synthesis just before reperfusion may contribute to the preservation of BBB, consistent with the suppression of MMP-9 expression and NOs inhibition (Gursoy-Ozdemir et al., 2000; Mark et al., 2004). MMPs mediated degradation of collagen and laminins in basal lamina disrupts the integrity of the vascular wall (Asahi et al.,

2001; Rosenberg and Navratil, 1997). Increases in MMP-2 and MMP-9 activities after focal cerebral ischemia may contribute to disruption of the BBB (Zhang et al., 2001). The present study indicated serious injuries, as well as increased MMP-9 expression, NO contents and NOs activities, in and around cortical microvessels after transient global cerebral ischemia. Crocin pretreatment repaired the vascular injuries, inhibited MMP-9 expression and reduced NO contents and NOs activities. What, then, may be the mechanism by which crocin protects the brain against this reperfusion injury? Phosphorylation of ERK localizes in areas of increased superoxide anion production after transient focal cerebral ischemia in mice. Rapid activation of ERK during reperfusion is likely a response to the generation of ROS (Noshita et al., 2002). In astrocytes, PKC and cytokine pathways triggered ERK activities is essential for MMP-9 upregulation (Arai et al., 2003). Further studies reported that not only do G-protein and β-arrestin contribute to the overall ERK signal but also that ERK participates in a feedback regulatory effects to GRK2 (Ahn et al., 2004). To further investigate the antioxidant mechanism of crocin, we detected the phosphorylation of ERK1/2 in cerebral microvessels. The results from the present study indicated that crocin pretreatment in vivo significantly inhibited the phosphorylation of ERK1/2 in cortical microvascular homogenates following transient global cerebral ischemia. The results provide further support that crocin has an important contribution to the ischemic BBB, which is due to the inhibition of ERK pathway and ultimate downregulation of MMPs. Furthermore, it was also indicated that global cerebral ischemia–reperfusion increased membrane (functional) GRK distribution in cortical microvessels in the present study. As is well known, GRKs are one of the most important mediators of G protein-coupled receptors (GPCRs) (Premont, 2005). The canonical model for GPCR desensitization involves GRKdependent receptor phosphorylation to promote the binding of arrestin proteins that block receptor–G protein interactions. To date, six different GRKs have been identified. GRK2, GRK3, and GRK5 target beta-adrenergic receptors (β-ARs) and are highly expressed in the brain (Lefkowitz et al., 1998). GRK2 and GRK3 are also known as β-AR kinases 1 (β-ARK-1) and 2 (βARK-2), respectively (Gagnon et al., 1998). β1-AR-mediated signals play an important role in vascular functional recovery in ischemic injury, ageing and hypertension. These findings are in accordance with studies reporting that GRK2 expression increased in hypertension, heart failure and ageing (Akhter et al., 2006; Gros et al., 2000; Ungerer et al., 1994) (Harris et al., 2001; Hata et al., 2006; Liu et al., 2005). Little is known about the mechanisms involved in the regulation of intracellular GRK2 levels. The GRK2 promoter may be activated after the stimulation of alpha 1-adrenergic receptors (α1-AR). However, stimulation of β-AR or chemokine receptors results in increased degradation of endogenous GRK2 (Penela et al., 2001; Ramos-Ruiz et al., 2000). At 2 h after focal ischemia, the translocation of GRK2 from the cytosol to the membrane occurs in ischemic cerebral hemispheres with an increase in β-AR-like immunoreactivity in reactive astrocytes in vivo (Imura et al., 1999). Further evidence indicates that GRK2 levels (especially membrane GRK2 expression) can be increased by cytokines, as well as by oxidative stress (Cobelens et al., 2006; Fardoun et al., 2006; Hagen et al., 2003;

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91

Fig. 7 – Effects of crocin on GRK2 subcellular distribution in cortical microvessels. Upper panel: Immunoblots of GRK2 expression in cortical microvascular Homogenates which were determined as described in the text. Lower panel: Bar graphs show quantitative evaluation of GRK2 expression. I/R caused a decreased GRK2 expression in the soluble fraction (cytosol) and an increased GRK2 expression in the particulate fraction (membrane). Crocin treatment led to decreased GRK2 translocation from the cytosol to the membrane. Results were obtained from three independent experiments; only one representative experiment is shown. n = 3. Data are mean ± SD. Nonparametric test was used for statistical analysis. *p < 0.05 compared with I/R group. Lane 1: sham; lane 2: I/R model; lane 3: I/R + crocin (20 mg/kg); lane 4: I/R + crocin (10 mg/kg); lane 5: I/R + crocin (5 mg/kg).

Mak et al., 2002; Mark et al., 2004). The results from the present study indicate that transient global ischemia promotes translocation of GRK2 from cytosol to membrane. The effect of crocin on GRK2 subcellular distribution indicates that GRK2 maybe one of the most important targets of crocin. In conclusion, crocin exhibits markedly protective effects in a mice model of transient global ischemia, which appears to involve the inhibition of reperfusion-induced oxidative/nitrative injury, ERK pathway activation and GRK translocation in the ischemic brain.

4.

Experimental procedures

4.1.

Extraction of saffron and isolation of crocin

4.2.

Male C57BL/6J mice, aged 12 weeks, were provided by the Department of Animal, Health Science Center of Peking University, were housed in the laboratory animal room and maintained at 25 ± 1 °C with 65 ± 5% humidity on a 12-h light/ dark cycle (lights on from 07:30 to 19:30) for at least 1 week before the start of the experiments. C57BL/6J mice were given food and water ad libitum. All experimental protocols described in this study were approved by the Ethics Review Committee for Animal Experimentation of Xiyuan Hospital, China Academy of Chinese Medical Sciences.

4.3.

Saffron was extracted thrice with 25× 60% alcohol at 80 °C, filtered and evaporated in a rotary vacuum evaporator. The extract was suspended 4× with water and the suspended extract was applied to an AB-8 macroporous adsorptive resin column chromatography and eluted with water and alcohol (20% → 70%). These isolated compounds concentrated from 70% alcohol were identified to be the total glycoside of crocin. Then the compounds were purified with a preparation liquid phase and eluted with a mobile phase containing water/ MeOH (1:1). The epicardium citri amorphous powder were concentrated from eluted fluids by decompression recovery and, through cryodesiccation, were identified to be crocin (purity = 99.9%) by comparing their physicochemical data with those in the literature, respectively. Crocin used in the present study was dissolved in distilled water.

Animals

Surgical operation and drug treatment

Before surgical operation, animals were divided into 5 groups randomly. From day 0 to day 21, mice were given crocin (20, 10, 5 mg/kg) intragastrically. The sham and I/R controls were given an equal volume of vehicle. BCCAO was carried out as described previously (Zhua et al., 2006). The animals were anesthetized by intraperitoneal (i.p.) injection of sodium pentobarbital (50 mg/kg) at day 20, and both common carotid arteries were carefully separated from the cervical sympathetic and vagal nerves through a ventral cervical incision. The bilateral common carotid arteries were exposed, isolated and clamped for 20 min with blood withdrawal through a tail–artery cannula to exsanguinate 0.5 ml blood. Blood pressure during ischemia and reperfusion was monitored as previously described (Yang et al., 2000). Body temperature was monitored with a rectal probe and maintained at 37.0–37.8 °C using a heat lamp. After blood

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reinfusion, clamp removal and wound closure, animals were placed in recovery cages with ambient temperature maintained at 22 °C. Animals that received the same surgical operation but without occlusion of the carotid arteries served as sham-operated controls.

4.4.

Transmission electron microscopy (TEM)

TEM was used to evaluate the effect of crocin on CMEC at the ultrastructural level. Mice were sacrificed on day 21. Cortical capillary fragments were fixed with 2.5% glutaraldehyde solution overnight at 4 °C and were washed with PBS after fixing with 1% osmic acid for 2 h. Being embedded in an Epon/ Araldite mixture and stained with uranyl acetate and lead citrate, the cortical microvessels were observed under a 1230 type transmission electron microscope (Electron Co., Japan) and photographed.

4.5. Measurement of NOs, SOD and GSH-px activities, NO and MDA levels in brain microvascular homogenates Mice were sacrificed on day 21. Mice brain capillary fragments were isolated using modified methods introduced by Abbott and Lin (Abbott et al., 1992; Lin and Rui, 1994). Briefly, fresh brain hemispheres were dropped into ice-cold Buffer A (10 mM HEPES, 11.9 mM NaHCO3, 140 mM NaCl, 10 mM KCl, 0.1% BSA), and the cerebellum, brain stem, choroid plexus, and meninges were carefully removed. The cortices were chopped with a scalpel for <1 min into uniform 2–3 mm pieces in Buffer A, digested in a collagenase/dispase solution (Ca, Mg-free HBSS with 20 units/ml DNase I) for 1 h at 37 °C to separate microvessels from other components, and the solution was then centrifuged in 15% dextran at 4500×g, at 4 °C for 20 min. The pellet containing crude microvessels was resuspended in PBS and was diluted into 1 mg protein/ml. Protein content was determined with BSA as a standard according to Bradford (1976) and the homogenates were used immediately for the assays of NOs, NO, MDA, GSH-px and SOD. NOs activity was measured by the L-arginine to NO conversion assay using a nitric oxide synthase assay kit (Jiancheng Institute of Biotechnology, Nanjing, China) according to the manufacturer's instructions. NO production was measured as nitrite (a stable metabolite of NO) concentrations using Griess reagent system following Jiancheng Institute of Biotechnology's protocols. MDA levels and SOD and GSH-px activities were determined following the kit instructions (Jiancheng Institute of Biotechnology). In brief, MDA was determined by the thiobarbituric acid method (Shimizu et al., 1999). The assays for total SOD and GSH-px were based on their ability to inhibit the oxidation of oxyamine by the xanthine–xanthine oxidase system.

4.6.

Western blotting

Mice cortical microvascular homogenates were collected as described above. The homogenate was centrifuged for 4 min at 3000×g and the resulting supernatants spun at 20,000×g for 30 min. Supernatants obtained include the cytoplasm protein and were diluted into 0.5 mg protein/ml for the measurement

of GRK2 and MMP-9 expression and ERK phosphorylation. Membrane pellets obtained were suspended in lysis buffer (240 mM NaCl, 2 mM EDTA, 2 mM leupeptin, 2 mM PMSF, pH 7.4) to yield about 0.5 mg protein/ml for GRK2 detection. Protein content was determined with BSA as a standard according to Bradford (1976). Proteins samples (20 μg/lane) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred to PVDF membranes (Millipore, Billerica, MA) through electroblotting. Blots were stained with GRK2 (Sc-562) antibody (Santa Cruz Biotechnology, Santa Cruz, CA), MMP-9 antibody, ERK1/2 and phosphoERK1/2 (pThr202/pTyr204) antibodies (Sigma Chemical Co, St. Louis, MO). Then the blots were developed by enhanced chemiluminescence using SuperSignal west femto maximum sensitivity substrate (Pierce, Rockford, IL) and Kodak X-AR film. Hema GSM-3.0 gel graph analyzing system was used to calculate the numerical value of every blot, and mean densitometric × areal values were depicted as bar graphs. All the experiments reported in this study were performed three times and the results were reproducible.

4.7.

Statistical analyses

Data were expressed as mean ± SD. The analysis of variance (ANOVA), regression–linear test and nonparametric test were used in the program SigmaStat (SPSS Software Products, Chicago, IL, USA) to determine significant differences among the groups. P values less than 0.05 were considered significant. Western blots were repeated at least three times for each sample and subjected to semiquantitative analysis to ensure maximal accuracy of the conclusion drawn from these data.

Acknowledgment This work was supported by the National Science Foundation for Post-doctor Scientists of China (No. 20060390584).

REFERENCES

Abbott, N.J., Hughes, C.C., Revest, P.A., Greenwood, J., 1992. Development and characterisation of a rat brain capillary endothelial culture: towards an in vitro blood-brain barrier. J. Cell. Sci. 103, 23–37. Abdullaev, F.I., 1993. Biological effects of saffron. Biofactors 4, 83–86. Ahn, S., Shenoy, S.K., Wei, H., Lefkowitz, R.J., 2004. Differential kinetic and spatial patterns of beta-arrestin and G protein-mediated ERK activation by the angiotensin II receptor. J. Biol. Chem. 279, 35518–35525. Akhter, S.A., D'Souza, K.M., Petrashevskaya, N.N., Mialet-Perez, J., Liggett, S.B., 2006. Myocardial beta1-adrenergic receptor polymorphisms affect functional recovery after ischemic injury. Am. J. Physiol.: Heart Circ. Physiol. 290, H1427–H1432. Arai, K., Lee, S.R., Lo, E.H., 2003. Essential role for ERK mitogen-activated protein kinase in matrix metalloproteinase-9 regulation in rat cortical astrocytes. Glia 43, 254–264. Asahi, M., Wang, X., Mori, T., Sumii, T., Jung, J.C., Moskowitz, M.A., Fini, M.E., Lo, E.H., 2001. Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood–brain barrier and

BR A IN RE S E A RCH 1 1 38 ( 20 0 7 ) 8 6 –9 4

white matter components after cerebral ischemia. J. Neurosci. 21, 7724–7732. Bradford, M.M., 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–524. Chan, P.H., 2001. Reactive oxygen radicals in signaling and damage in the ischemic brain. J. Cereb. Blood Flow Metab. 21, 2–14. Cobelens, P.M., Kavelaars, A., Heijnen, C.J., Ribas, C., Mayor Jr., F., Penela, P., 2006. Hydrogen peroxide impairs GRK2 translation via a calpain-dependent and cdk1-mediated pathway. Cell Signalling. Collino, M., Aragno, M., Mastrocola, R., Benetti, E., Gallicchio, M., Dianzani, C., Danni, O., Thiemermann, C., Fantozzi, R., 2006. Oxidative stress and inflammatory response evoked by transient cerebral ischemia/reperfusion: effects of the PPAR-alpha agonist WY14643. Free Radical Biol. Med. 41, 579–589. Crack, P.J., Taylor, J.M., 2005. Reactive oxygen species and the modulation of stroke. Free Radical Biol. Med. 38, 1433–1444. del Zoppo, G.J., 2006. Stroke and neurovascular protection. N. Engl. J. Med. 354, 553–555. del Zoppo, G.J., von Kummer, R., Hamann, G.F., 1998. Ischaemic damage of brain microvessels: inherent risks for thrombolytic treatment in stroke. J. Neurol., Neurosurg. Psychiatry 65, 1–9. Fagan, S.C., Hess, D.C., Hohnadel, E.J., Pollock, D.M., Ergul, A., 2004. Targets for vascular protection after acute ischemic stroke. Stroke 35, 2220–2225. Fardoun, R.Z., Asghar, M., Lokhandwala, M., 2006. Role of oxidative stress in defective renal dopamine D1 receptor–G protein coupling and function in old Fischer 344 rats. Am. J. Physiol., Renal Fluid Electrolyte Physiol. 291, F945–F951. Gagnon, A.W., Kallal, L., Benovic, J.L., 1998. Role of clathrin-mediated endocytosis in agonist-induced down-regulation of the beta2-adrenergic receptor. J. Biol. Chem. 273, 6976–6981. Gros, R., Chorazyczewski, J., Meek, M.D., Benovic, J.L., Ferguson, S.S., Feldman, R.D., 2000. G-Protein-coupled receptor kinase activity in hypertension: increased vascular and lymphocyte G-protein receptor kinase-2 protein expression. Hypertension 35, 38–42. Gursoy-Ozdemir, Y., Bolay, H., Saribas, O., Dalkara, T., 2000. Role of endothelial nitric oxide generation and peroxynitrite formation in reperfusion injury after focal cerebral ischemia. Stroke 31, 1974–1980 discussion 1981. Gursoy-Ozdemir, Y., Can, A., Dalkara, T., 2004. Reperfusion-induced oxidative/nitrative injury to neurovascular unit after focal cerebral ischemia. Stroke 35, 1449–1453. Hagen, S.A., Kondyra, A.L., Grocott, H.P., El-Moalem, H., Bainbridge, D., Mathew, J.P., Newman, M.F., Reves, J.G., Schwinn, D.A., Kwatra, M.M., 2003. Cardiopulmonary bypass decreases G protein-coupled receptor kinase activity and expression in human peripheral blood mononuclear cells. Anesthesiology 98, 343–348. Harris, C.A., Chuang, T.T., Scorer, C.A., 2001. Expression of GRK2 is increased in the left ventricles of cardiomyopathic hamsters. Basic Res. Cardiol. 96, 364–368. Hata, J.A., Williams, M.L., Schroder, J.N., Lima, B., Keys, J.R., Blaxall, B.C., Petrofski, J.A., Jakoi, A., Milano, C.A., Koch, W.J., 2006. Lymphocyte levels of GRK2 (betaARK1) mirror changes in the LVAD-supported failing human heart: lower GRK2 associated with improved beta-adrenergic signaling after mechanical unloading. J. Card. Fail. 12, 360–368. He, S.Y., Qian, Z.Y., Tang, F.T., Wen, N., Xu, G.L., Sheng, L., 2005. Effect of crocin on experimental atherosclerosis in quails and its mechanisms. Life Sci. 77, 907–921. Hosseinzadeh, H., Sadeghnia, H.R., 2005. Safranal, a constituent of Crocus sativus (saffron), attenuated cerebral ischemia induced oxidative damage in rat hippocampus. J. Pharm. Pharm. Sci. 8, 394–399.

93

Huang, Y., McNamara, J.O., 2004. Ischemic stroke: “acidotoxicity” is a perpetrator. Cell 118, 665–666. Huang, Z., Huang, P.L., Panahian, N., Dalkara, T., Fishman, M.C., Moskowitz, M.A., 1994. Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science 265, 1883–1885. Imura, T., Shimohama, S., Sato, M., Nishikawa, H., Madono, K., Akaike, A., Kimura, J., 1999. Differential expression of small heat shock proteins in reactive astrocytes after focal ischemia: possible role of beta-adrenergic receptor. J. Neurosci. 19, 9768–9779. Kakita, T., Suzuki, M., Takeuchi, H., Unno, M., Matsuno, S., 2002. Significance of xanthine oxidase in the production of intracellular oxygen radicals in an in-vitro hypoxia–reoxygenation model. J. Hepatobiliary Pancreat. Surg. 9, 249–255. Kim, G.W., Kondo, T., Noshita, N., Chan, P.H., 2002. Manganese superoxide dismutase deficiency exacerbates cerebral infarction after focal cerebral ischemia/reperfusion in mice: implications for the production and role of superoxide radicals. Stroke 33, 809–815. Kondo, T., Reaume, A.G., Huang, T.T., Murakami, K., Carlson, E., Chen, S., Scott, R.W., Epstein, C.J., Chan, P.H., 1997. Edema formation exacerbates neurological and histological outcomes after focal cerebral ischemia in CuZn-superoxide dismutase gene knockout mutant mice. Acta Neurochir., Suppl. 70, 62–64. Kontos, H.A., 2001. Oxygen radicals in cerebral ischemia: the 2001 Willis lecture. Stroke 32, 2712–2716. Lee, I.A., Lee, J.H., Baek, N.I., Kim, D.H., 2005. Antihyperlipidemic effect of crocin isolated from the fructus of Gardenia jasminoides and its metabolite crocetin. Biol. Pharm. Bull. 28, 2106–2110. Lefkowitz, R.J., Pitcher, J., Krueger, K., Daaka, Y., 1998. Mechanisms of beta-adrenergic receptor desensitization and resensitization. Adv. Pharmacol. 42, 416–420. Lin, A.Y., Rui, Y.C., 1994. Platelet-activating factor induced calcium mobilization and phosphoinositide metabolism in cultured bovine cerebral microvascular endothelial cells. Biochim. Biophys. Acta. 1224, 323–328. Liu, S., Premont, R.T., Kontos, C.D., Zhu, S., Rockey, D.C., 2005. A crucial role for GRK2 in regulation of endothelial cell nitric oxide synthase function in portal hypertension. Nat. Med. 11, 952–958. Madl, C., Holzer, M., 2004. Brain function after resuscitation from cardiac arrest. Curr. Opin. Crit. Care 10, 213–217. Maier, C.M., Hsieh, L., Crandall, T., Narasimhan, P., Chan, P.H., 2006. Evaluating therapeutic targets for reperfusion-related brain hemorrhage. Ann. Neurol. 59, 929–938. Mak, J.C., Hisada, T., Salmon, M., Barnes, P.J., Chung, K.F., 2002. Glucocorticoids reverse IL-1beta-induced impairment of beta-adrenoceptor-mediated relaxation and up-regulation of G-protein-coupled receptor kinases. Br. J. Pharmacol. 135, 987–996. Mark, K.S., Burroughs, A.R., Brown, R.C., Huber, J.D., Davis, T.P., 2004. Nitric oxide mediates hypoxia-induced changes in paracellular permeability of cerebral microvasculature. Am. J. Physiol.: Heart Circ. Physiol. 286, H174–H180. Noshita, N., Sugawara, T., Hayashi, T., Lewen, A., Omar, G., Chan, P.H., 2002. Copper/zinc superoxide dismutase attenuates neuronal cell death by preventing extracellular signal-regulated kinase activation after transient focal cerebral ischemia in mice. J. Neurosci. 22, 7923–7930. Ochiai, T., Ohno, S., Soeda, S., Tanaka, H., Shoyama, Y., Shimeno, H., 2004. Crocin prevents the death of rat pheochromyctoma (PC-12) cells by its antioxidant effects stronger than those of alpha-tocopherol. Neurosci. Lett. 362, 61–64. Penela, P., Elorza, A., Sarnago, S., Mayor Jr., F., 2001. Beta-arrestin- and c-Src-dependent degradation of G-protein-coupled receptor kinase 2. EMBO J. 20, 5129–5138.

94

BR A IN RE S EA RCH 1 1 38 ( 20 0 7 ) 8 6 –94

Premkumar, K., Abraham, S.K., Santhiya, S.T., Ramesh, A., 2003. Protective effects of saffron (Crocus sativus Linn.) on genotoxins-induced oxidative stress in Swiss albino mice. Phytother. Res. 17, 614–617. Premont, R.T., 2005. Once and future signaling: G protein-coupled receptor kinase control of neuronal sensitivity. Neuromol. Med. 7, 129–147. Preston, E., Webster, J., 2002. Differential passage of [14C]sucrose and [3H]inulin across rat blood–brain barrier after cerebral ischemia. Acta Neuropathol. (Berl.) 103, 237–242. Preston, E., Webster, J., 2004. A two-hour window for hypothermic modulation of early events that impact delayed opening of the rat blood–brain barrier after ischemia. Acta Neuropathol. (Berl.) 108, 406–412. Preston, E., Sutherland, G., Finsten, A., 1993. Three openings of the blood–brain barrier produced by forebrain ischemia in the rat. Neurosci. Lett. 149, 75–78. Ramos-Ruiz, R., Penela, P., Penn, R.B., Mayor Jr., F., 2000. Analysis of the human G protein-coupled receptor kinase 2 (GRK2) gene promoter: regulation by signal transduction systems in aortic smooth muscle cells. Circulation 101, 2083–2089. Rosenberg, G.A., Navratil, M., 1997. Metalloproteinase inhibition blocks edema in intracerebral hemorrhage in the rat. Neurology 48, 921–926. Ryter, S.W., Choi, A.M., 2005. Heme oxygenase-1: redox regulation of a stress protein in lung and cell culture models. Antioxid. Redox Signal. 7, 80–91. Saleem, S., Ahmad, M., Ahmad, A.S., Yousuf, S., Ansari, M.A., Khan, M.B., Ishrat, T., Islam, F., 2006. Effect of saffron (Crocus sativus) on neurobehavioral and neurochemical changes in cerebral ischemia in rats. J. Med. Food 9, 246–253. Schaller, B., 2005. Prospects for the future: the role of free radicals in the treatment of stroke. Free Radical Biol. Med. 38, 411–425. Shah, Z.A., Gilani, R.A., Sharma, P., Vohora, S.B., 2005. Cerebroprotective effect of Korean ginseng tea against global and focal models of ischemia in rats. J. Ethnopharmacol. 101, 299–307. Shimizu, I., Ma, Y.R., Mizobuchi, Y., Liu, F., Miura, T., Nakai, Y., Yasuda, M., Shiba, M., Horie, T., Amagaya, S., Kawada, N., Hori, H., Ito, S., 1999. Effects of Sho-saiko-to, a Japanese herbal medicine, on hepatic fibrosis in rats. Hepatology 29, 149–160.

Sugiura, M., Shoyama, Y., Saito, H., Abe, K., 1994. Crocin (crocetin di-gentiobiose ester) prevents the inhibitory effect of ethanol on long-term potentiation in the dentate gyrus in vivo J. Pharmacol. Exp. Ther. 271, 703–707. Ungerer, M., Parruti, G., Bohm, M., Puzicha, M., DeBlasi, A., Erdmann, E., Lohse, M.J., 1994. Expression of beta-arrestins and beta-adrenergic receptor kinases in the failing human heart. Circ. Res. 74, 206–213. Weigl, M., Tenze, G., Steinlechner, B., Skhirtladze, K., Reining, G., Bernardo, M., Pedicelli, E., Dworschak, M., 2005. A systematic review of currently available pharmacological neuroprotective agents as a sole intervention before anticipated or induced cardiac arrest. Resuscitation 65, 21–39. White, B.C., Sullivan, J.M., DeGracia, D.J., O'Neil, B.J., Neumar, R.W., Grossman, L.I., Rafols, J.A., Krause, G.S., 2000. Brain ischemia and reperfusion: molecular mechanisms of neuronal injury. J. Neurol. Sci. 179, 1–33. Yang, G., Chan, P.H., Chen, J., Carlson, E., Chen, S.F., Weinstein, P., Epstein, C.J., Kamii, H., 1994. Human copper–zinc superoxide dismutase transgenic mice are highly resistant to reperfusion injury after focal cerebral ischemia. Stroke 25, 165–170. Yang, G., Kitagawa, K., Ohtsuki, T., 2000. Regional difference of neuronal vulnerability in the murine hippocampus after transient forebrain ischemia. Brain. Res. 870, 195–198. Yoshida, T., Waeber, C., Huang, Z., Moskowitz, M.A., 1995. Induction of nitric oxide synthase activity in rodent brain following middle cerebral artery occlusion. Neurosci. Lett. 194, 214–218. Zhang, Y., Shoyama, Y., Sugiura, M., Saito, H., 1994. Effects of Crocus sativus L. on the ethanol-induced impairment of passive avoidance performances in mice. Biol. Pharm. Bull. 17, 217–221. Zhang, Z.G., Zhang, L., Tsang, W., Goussev, A., Powers, C., Ho, K.L., Morris, D., Smyth, S.S., Coller, B.S., Chopp, M., 2001. Dynamic platelet accumulation at the site of the occluded middle cerebral artery and in downstream microvessels is associated with loss of microvascular integrity after embolic middle cerebral artery occlusion. Brain Res. 912, 181–194. Zhua, Y., Saito, K., Murakamia, Y., Asano, M., Iwakura, Y., Seishima, M., 2006. Early increase in mRNA levels of proinflammatory cytokines and their interactions in the mouse hippocampus after transient global ischemia. Neurosci. Lett. 393, 122–126.