Lipoxin A4 analogue protects brain and reduces inflammation in a rat model of focal cerebral ischemia reperfusion

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Research Report

Lipoxin A4 analogue protects brain and reduces inflammation in a rat model of focal cerebral ischemia reperfusion Xi-Hong Yea , Yan Wub , Pei-Pei Guoa , Jie Wanga , Shi-Ying Yuanc , You Shangc,⁎, Shang-Long Yaoa,⁎ a

Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China c Department of Critical Care, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China b

A R T I C LE I N FO

AB S T R A C T

Article history:

Inflammation, which is known to be detrimental to the neurological outcome during the

Accepted 28 January 2010

acute phase after ischemia, provides a potential preventative or therapeutic approach for

Available online 4 February 2010

acute stroke. Lipoxins are endogenous lipoxygenase derived eicosanoids and evokes protective actions in a range of pathophysiologic processes. Here, we evaluated the

Keywords:

efficacy of 5 (S), 6 (R)-lipoxin A4 methyl ester (LXA4 ME), a stable synthetic analogue of lipoxin

Lipoxin A4 methyl ester

A4 in cerebral ischemia reperfusion injury in rats. Transient focal cerebral ischemia was

Cerebral ischemia reperfusion

induced by middle cerebral artery occlusion for 2 h. Intracerebroventricular administration

Anti-inflammation

of LXA4 ME immediately after onset of ischemia ameliorated neurological dysfunctions,

Cytokines

reduced infarction volume and attenuated neuronal apoptosis. Moreover, Treatment with

Glia activation

LXA4 ME suppressed neutrophils infiltration and lipid peroxidation levels; inhibited the activation of microglia and astrocytes; reduced the expression of pro-inflammatory cytokines TNF-α and IL-1β; and up-regulated the expression of anti-inflammatory cytokines IL-10 and TGF-β1 in the ischemic brain. In addition, activation of NF-κΒ was inhibited by LXA4 ME treatment. These results demonstrate that treatment of LXA4 ME affords strong neuroprotective effect against cerebral ischemia reperfusion injury, and that these effects might be associated with its anti-inflammatory property. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

Ischemic stroke is one of the significant causes of morbidity and mortality in the world. Although different mechanisms

are involved in the pathogenesis of stroke, inflammatory response occurs after stroke and contributes to ischemic brain injury. After ischemia onset, inflammatory cells such as neutrophils and glia take part in inflammatory response.

⁎ Corresponding authors. Y. Shang is to be contacted at Department of Critical Care, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1277 Jiefangdadao, Wuhan, 430022 China. Fax: +86 27 85351660. S.-L. Yao, Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, No.1277 Jiefangdadao, Wuhan, 430022 China. E-mail addresses: [email protected] (Y. Shang), [email protected] (S.-L. Yao). Abbreviations: LX, Lipoxin; LXA4 ME, LXA4 methyl ester (5S,6R,15S-trihydroxyl-7,9,13-trans-11-cis-eicosatetraenoic acid methyl ester); I/ R, ischemia/reperfusion; MCAO, middle cerebral artery occlusion; MPO, myeloperoxidase; MDA, malondialdehyde; IL-1β/10, interleukin1β/10; TNF-α, tumor necrosis factor-α; TGF-β1, transforming growth factor-β1; GFAP, glial fibrillary acidic protein; PPARγ, peroxisome proliferator-activated receptor γ; TTC, 2,3,5-triphenyltetrazolium chloride; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; NF-κB, nuclear factor-kappa B; EMSA, electrophoretic mobility shift assay 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.01.079

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Moreover, cytokines such as IL-1β, TNF-α, IL-10 and TGF-β1 are also important mediators of the inflammatory reactions in cerebral ischemia. Blocking various aspects of the inflammatory cascade has been shown to ameliorate injury in experimental stroke (Durukan and Tatlisumak, 2007; Amantea et al., 2009; Huang et al., 2006; Wang et al., 2007; Han and Yenari, 2003; Allan and Rothwell, 2001). Thus, target inflammation followed cerebral ischemia would be an ideal preventative or therapeutic approach for neuroprotection. Lipoxins are recently discovered kinds of endogenous anti-inflammatory lipid-based autacoids, which generated from arachidonic acid via lipoxygenase-mediated transcellular biosynthesis. Lipoxin A4 (LXA4) is one of the principal LXs generated by mammalian cells. LXA4 binds to its specific G protein-coupled receptor termed ALXR (Chiang et al., 2005), which can be cloned in neutrophils, monocytes, macrophage as well as resident cells such as astrocytes, microglia and neural stem cells in central nervous system, suggesting that these cells may be targets for LXs action in the brain. (Maddox et al., 1997; SodinSemrl et al., 2004; Svensson et al., 2007; Wada et al., 2006). As LXs are biosynthesized and rapidly enzymically inactivated, stable and more potent analogues were constructed (Serhan et al., 1995; Chiang et al., 2000). Native LXs and their stable analogues including LXA4 methyl ester (LXA4 ME), exert their biological effects on a number of major inflammatory disorders, including vascular injury, kidney disease, periodontal disease, asthma, arthritis and cystic fibrosis (Petasis et al., 2008). LXs are considered as endogenous ‘stop signals’ for inflammation, which can downregulate or counteract the formation and action of pro-inflammatory mediators and promote resolution (Serhan and Oliw, 2001). Therefore, it may be a novel strategy that mimicking the action of endogenous anti-inflammatory and pro-resolution lipid mediators to reduce cerebral inflammation. The present study was undertaken aimed to explore the neuroprotective effects of LXA4 ME on ischemic brain and discover its underlying mechanisms.

2.

Results

2.1.

LXA4 ME ameliorated neurological dysfunctions

Neurologic deficits, evaluated at 24 h after reperfusion, were significantly increased in I/R group compared with LXA4 ME group (neurological scores 2.61 ± 0.37 versus 1.22 ± 0.21, p < 0.01, n = 12, respectively, Fig. 1). No rats appeared with neurological impairment symptoms in the sham group.

2.2.

LXA4 ME reduced brain infarction

Representative coronal brain sections from the experiment groups were showed (Fig. 2A). Infarct volume at 24 h after reperfusion decreased significantly in LXA4 ME group compared with I/R group. The infarct size was quantified by the ratio of corrected infarcted volume to whole brain volume (Fig. 2B).

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Fig. 1 – Effect of LXA4 ME on neurological deficit scores after I/R in rats. Elevated neurological deficit scores after I/R were significantly attenuated by LXA4 ME (0.3 nM). Bars represent mean ± SEM (n = 12); *p < 0.01 versus sham group and #p < 0.01 versus I/R group.

2.3.

LXA4 ME extenuated pathological damage in the cortex

Hematoxylin and eosin staining was showed in Fig. 3. In the sham group no neuronal damage observed and no inflammatory cells infiltrated. Neurons were eumorphism with normal cellular architecture. The nuclei were in the cell center and clear stained. In I/R group, the brain tissue characterized by a majority of degenerated and necrotic cells, which contained pyknotic nuclei, cavitation with neuronal loss and disorders. In LXA4 ME group, there was moderate neuronal damage in the ischemic area.

2.4.

LXA4 ME attenuated neuronal apoptosis

There were no TUNEL-positive cells detected in the right cortex of sham group. However, in the ischemic cortex of I/R group large number of TUNEL-positive cells were observed. In LXA4 ME group the number of TUNEL-positive cells decreased significantly compared with I/R group, as shown in Fig. 4.

2.5. LXA4 ME inhibited glia activation and neutrophil infiltration Immunohistochemistry staining for GFAP and OX42 were used to assess astrocyte and microglia respectively. In the sham group, there were a few GFAP-positive cells detected in the right cortex. In I/R group there were large number of GFAPpositive cells observed in the ischemic cortex. In LXA4 ME group the number of GFAP-positive cells was less than in I/R group (Fig. 5). As previously reported, the morphology of the microglia in the ischemia cortex appears more bipolar rather than stellate (Barron, 1995). Few microglia (OX42 immunostaining positive cell) could be found in the sham group. In I/R group large number of OX42-positive cell could be found, but in LXA4 ME group the number of OX42-positive cells decreased (Fig. 6). After 24 h of reperfusion, MPO activities in the ischemic cortex were higher in I/R group than in sham group. There was significant reduction of MPO activities in LXA4 ME group compared with I/R group, which indicated LXA4 ME inhibited neutrophils accumulation (Fig. 7A).

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2.7.

LXA4 ME downregulated pro-inflammatory cytokines

The time course changes in concentration of IL-1β and TNF-α were measured by ELISA in the ischemic cortex at 0 h, 6 h, 12 h and 24 h of reperfusion after MCAO. The TNF-α levels were significantly increased as early as 0–6 h after reperfusion, declined close to sham group level by 24 h. A similar temporal pattern of increases in IL-1β concentration was also observed. The IL-1β levels were also elevated by 0 h after reperfusion and the level were maintained from 6 h to 12 h, followed by a slightly decline at 24 h. The concentration of these factors was significantly higher in I/R group than in LXA4 ME group (Fig. 8).

2.8.

LXA4 ME up-regulated anti-inflammatory cytokines

The time course changes in concentration of IL-10 and TGF-β1 were investigated in the ischemic cortex after I/R injury (0–24 h). The IL-10 levels increased from 0 h to 12 h after reperfusion and slightly declined at 24 h, which showed a peak induction at 6 h. But, in LXA4 ME group the peak appeared at 24 h. There is significant difference of IL-10 concentration between I/R group and LXA4 ME group. A similar trend in TGF-β1 levels was also observed between the two groups (Fig. 9).

2.9. LXA4 ME inhibited nuclear factor-κΒ (NF-κΒ) activation As shown in Fig. 10, NF-κΒ activity of the nuclear extracts of ischemic cortex was measured by western blotting. NF-κΒ activity was induced by ischemia reperfusion injury at 24 h of reperfusion, but suppressed by LXA4 ME treatment. Activity of NF-κΒ/DNA binding was analyzed by electrophoretic mobility shift assay (EMSA). DNA binding activity of NF-κΒ was present at very low level in sham group, but significantly increased in I/R group. DNA binding activity of NF-κΒ was suppressed in LXA4 ME group compared with I/R group. Fig. 2 – Effect of LXA4 ME on cerebral infarct volume after I/R in rats. (A) Representative TTC stained brain sections were shown where rats were subjected to 2 h ischemia followed by 24 h reperfusion (I/R) and intracerebroventricular injection with LXA4 ME (0.3 nM). White is infarct area and red is normal area. Compared with I/R group, LXA4 ME can restrain the brain injury induced by ischemia/reperfusion insult, which manifested by the significantly decrease white color area. (B) Quantification of infarct volume at 24 h. The ratio of corrected infarct volume to whole brain volume was calculated for the cerebral infarct size. Infarct volume was decreased at 24 h with LXA4 ME treatment. Bars represent mean ± SEM (n = 6); *p < 0.01 versus sham and #p < 0.05 versus I/R.

2.6.

LXA4 ME reduced lipid peroxidation levels

At 24 h of reperfusion after MCAO, there was a significant increase of MDA levels in I/R group compared with sham group. However, MDA levels were lower in LXA4 ME group than in I/R group (Fig. 7B).

3.

Discussion

LXs are kinds of endogenous anti-inflammatory lipid-based autacoids, separated from the biosynthesis of prostaglandins and leukotrienes, endogenously produced in picogram to nanogram range in most other murine during inflammation and disease pathogenesis (Serhan and Savill, 2005; Parkinson, 2006). Previous studies showed LX analogues were active when administrated intravenously, intraperitoneal, topically and orally if available, which demonstrated effective even in the nanogram dose range in vivo. However, it is little known whether LXA4 ME could cross the blood brain barrier. It was reported spinal delivery of lipoxin A4, as well as stable analogues, attenuated inflammation-induced pain. In that report, rats received an injection of lipoxins in nanogram range intrathecally (0.3 nM) (Svensson et al., 2007). In our present research, the same dose of LXA4 ME was chosen and administrated by intracerebroventricular Injection. In the present study, we clearly demonstrated that LXA4 ME protected the brain against ischemia and reperfusion injury in a rat MCAO model. LXA4 ME significantly ameliorated neurological deficits, reduced cerebral infarct volume and

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Fig. 3 – HE staining in the cortex. Photomicrographs showing neuronal damage in the I/R and LXA4 ME group (magnification 400×).

attenuated neuronal apoptosis after MCAO. This protection is correlative with the anti-inflammation effect of LXA4 ME, which is based on the observation that LXA4 ME can inhibit inflammatory cells and inflammatory cytokines response to ischemia injury. Cerebral ischemia triggers complex cellular processes involving not only the recruitment of inflammatory blood cells but also the activation of resident glia (Wang et al., 2007). It is detrimental within several hours after ischemic insult, excess circulating neutrophils infiltrated into brain. Therapies that prevent the neutrophil infiltration during the acute phase after ischemia have been shown to be neuroprotective (Durukan and Tatlisumak, 2007; Huang et al., 2006; Wang et al., 2007). Astrocytes and microglia which activated and developed in the ischemic tissue, also play an important role in inflammatory response followed ischemia. Our results showed that treatment with LXA4 ME suppressed neutrophil infiltration and glia activation in the post-ischemic brain. It may contribute to the survival of neuron.

Since brain has large amount of unsaturated fatty acids, which are vulnerable to free radical-induced lipid peroxidation. The burst of oxygen radical not only in the early phases of reperfusion but also occurs in later periods (Kontos, 2001). We found that administration of LXA4 ME could reduce the level of lipid peroxidation, which indicated LXA4 ME can attenuate the generation of oxygen radicals. It is consistent with previous reports, LX could suppress the generation of reactive oxygen species (Nascimento-Silva et al., 2007; Zhou et al., 2007). Overexpression cytokines also play an important role in inflammatory response followed ischemia. Ischemia leads to rapid up-regulation of pro-inflammatory cytokines IL-1β and TNF-α. In previous studies, IL-1β levels increased very early following permanent MCAO and peaked within hours of reperfusion in transient focal ischemic models in rodents (Davies et al., 1999; Zhang et al., 1998). TNF-α was also upregulated in the brain after ischemia with similar expression pattern as IL-1β. These cytokines are supposed to play crucial roles in neutrophil infiltration and glia activation evoked by

Fig. 4 – Effect of LXA4 ME on neuron apoptosis. (A) Representative photomicrographs of TUNEL-positive cells (brown staining) in the MCA territory of the ischemic cortex (magnification 400×). (B) Quantification of TUNEL-positive cells at 24 h reperfusion. MCAO caused an increase of TUNEL-positive cells, which were significantly decreased by LXA4 ME. Bars represent mean ± SEM (n = 6); *p < 0.01 versus sham and #p < 0.05 versus I/R.

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Fig. 5 – LXA4 ME treatment reduces the number of GFAP-positive cell and in the ipsilateral brain hemisphere after I/R. The expression of GFAP (brown staining) in the cerebral ischemic hemisphere were detected by immunohistological assay. LXA4 ME can significantly inhibit the activation of astrocyte. (B) Quantification of GFAP-positive cell at 24 h of reperfusion.

cerebral ischemia, which exacerbate cerebral ischemia reperfusion injury. Researchers have shown that the injection of antagonists of IL-1β and TNF-α relieved the ischemia injury (Loddick and Rothwell, 1996; Barone et al., 1997). Our findings show that LXA4 ME treatment markedly suppresses the expression of pro-inflammatory cytokines IL-1β and TNF-α. This could explain the function that LXA4 ME can attenuate neutrophil infiltration and glia activation. The outcome of post-ischemic inflammatory processes mainly depends on the imbalance between the activation of pro-inflammatory cytokines cascade and the induction of

anti-inflammatory cytokines (Wang et al., 2007). Anti-inflammatory cytokines IL-10 and TGF-β1 have been implicated as key mediators for the recovery of ischemic stroke (Zhang et al., 1994; Pantoni et al., 1998). Previous reports have shown that both exogenous administration and gene transfer of IL-10 and TGF-β1 mediated neuroprotection after ischemic insult (Frenkel et al., 2003; Ooboshi et al., 2005; Pang et al., 2001). Moreover, the inhibitory effects of IL-10 on pro-inflammatory cytokines production have already been documented in various peripheral inflammatory models including endotoxemia, pancreatitis, or hepatitis (Moore et al., 2001). In the present study, LXA4

Fig. 6 – LXA4 ME treatment reduces the number of OX42-positive cell in the ipsilateral brain hemisphere after I/R. The expression of OX42 (brown staining) in the injured cerebral hemisphere were detected by immunohistological assay. LXA4 ME can significantly inhibit the activation of microglia. (B) Quantification of OX42-positive cell at 24 h of reperfusion.

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Fig. 7 – Effect of LXA4 ME on MPO activities and lipid peroxidation levels. (A) Assay of MPO activities in ischemic cortex. LXA4 ME treatment reduced MPO activities. (B) Assay of MDA content in ischemic cortex. LXA4 ME treatment inhibited the increase of MDA. Bars represent mean ± SEM (n = 6); *p < 0.05 versus sham and #p < 0.05 versus I/R.

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ME up-regulates the expression of IL-10 and TGF-β1, which indicating that LXA4 ME neuroprotection after MCAO may be partly mediated by these anti-inflammatory cytokines. NF-κB is known to be one of the crucial transcription factors required for maximal transcription of a wide array of pro-inflammatory molecules including TNF-α, IL-1β and other mediators. NF-κB released from IκB translocates into nucleus, where it enhanced the transcription of cytokines. The pivotal role of activation of NF-κB in inflammation during cerebral ischemia has been well elucidated in previous studies (Nurmi et al., 2004; Stephenson et al., 2000). In present study, cerebral ischemia reperfusion caused magnitude of NF-κB activation, which ran parallel with a marked increase in TNF-α and IL-1β. However, NF-κB activation in the ischemic tissue was suppressed significantly by LXA4 ME treatment followed by the decrease of TNF-α and IL-1β. These observations were concordance with reports in epithelial cells, which LXA4 activated ALX receptor and inhibited NF-κΒ activity (Medeiros et al., 2008), and in human leukocytes, which LXA4 and ATL inhibited NF-kappa B and AP-1 (József et al., 2002). In addition, LXs may also play a role through peroxisome proliferator-activated receptor γ (PPARγ) signaling pathway. Recently, Sobrado et al. demonstrated that LXA4 afforded neuroprotection, which was partly inhibited by the PPARγ antagonist T0070907, and increased PPARγ transcriptional activity in experiment stroke (Sobrado et al., 2009). These findings suggested LXA4 maybe exert neuroprotection through directly interfering with intracellular signaling pathways.

Fig. 8 – Time course of expression of pro-inflammatory cytokines in ischemic brain after MCAO and inhibitory effect of LXA4 ME. Cerebral tissue homogenates were obtained from the ischemic cortex and the content of IL-1β, and TNF-α were measured by ELISA, respectively. The concentration of IL-1β, and TNF-α were significantly increase after I/R with a peak at 6 h, which can be inhibited by LXA4 ME treatment. Bars represent mean ± SEM (n = 6); *p < 0.05 versus sham and #p < 0.05 versus I/R.

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Fig. 9 – Time course of expression of anti-inflammatory cytokines in ischemic brain after MCAO and up-regulated by LXA4 ME treatment. Cerebral tissue homogenates were obtained from the ischemic cortex and the content of IL-10 and TGF-β1 were measured by ELISA, respectively. The concentration of IL-10 and TGF-β1 were significantly increase after I/R compared to sham group, but declined at 24 h in I/R group. However there is the highest level at 24 h after LXA4 ME administered. Bars represent mean ± SEM (n = 6); *p < 0.05 versus sham and #p < 0.05 versus I/R.

In conclusion, these results suggest that LXA4 ME has a profound effect on attenuating focal ischemia induced inflammatory response at cellular and molecular level. The

underlying mechanism could be reducing the expression of pro-inflammatory cytokines TNF-α and IL-1β; and up-regulating the expression of anti-inflammatory cytokines IL-10 and

Fig. 10 – LXA4 ME inhibited NF-κΒ activation induced by 2 h ischemia and 24 h reperfusion. (A) Nuclear extracts were prepared from the tissues of the ischemic cortex for western blotting with NF-κΒ (p65) antibody. (B) Panels show representative immunoblots of NF-κΒ (p65) and β-actin in three groups ,*p < 0.05 versus sham and #p < 0.05 versus I/R. (C) NF-κΒ binding activity in ischemic cortex in three groups was assessed by EMSA.

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TGF-β1, inhibition of NF-κB activation. These findings will provide experimental and therapeutic options for the treatment of a broad range of pathologic disorders associated with brain ischemia.

4.4.

181

Measurement of infarct volume

All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals. All the usage and procedures were approved by the committee of experimental animals of Tongji Medical College. 96 adult male Sprague–Dawley rats weighting 200–250 g were randomized divided into three groups: (i) sham group: n = 12, no middle cerebral artery occlusion; (ii) I/R group: n = 42, MCAO ischemia and reperfusion; (iii) LXA4 ME group: n = 42, MCAO ischemia and reperfusion with 0.3 nM LXA4 ME (Cayman, Ann Arbor, MI, USA). The rats were available ad lib except under certain experimental conditions.

After 24 h of reperfusion, the rats were anesthetized and decapitated. Cerebral infarct volumes were measured as described previously (Swanson and Sharp, 1991). Briefly, brains (n = 6 per group) were cut into 2-mm thick coronal section in a cutting block, and were immediately immersed in 2% TTC (2,3,5-triphenyltetrazolium chloride) (Sigma, St. Louis, MO, USA) for 20 min at 37 °C ,followed by overnight immersion in 4% paraformaldehyde. The infarct area was demarcated and analyzed by Image J software (NIH Image, Version 1.61, Bethesda, USA). The demarcation between infarcted and noninfarcted tissue was outlined, and the total volume of infarction was calculated by integration of the lesion areas from all six sections measured. To compensate for the effect of brain edema, the corrected infarct volume was calculated: Corrected infarct area = Measured infarct area × {1 − [(Ipsilateral hemisphere area − Contralateral hemisphere area)/Contralateral hemisphere]} (Xing et al., 2008). The infarct size was quantified by the ratio of corrected infarcted volume to whole brain volume.

4.2.

4.5.

4.

Experimental procedures

4.1.

Animal models

Middle cerebral artery occlusion model

Rats were anesthetized with chloral hydrate (350 mg/kg, i.p.) and subjected to middle cerebral artery occlusion (MCAO) as described previously with slight modification (Longa et al., 1989). Briefly, the right common carotid artery, internal carotid artery (ICA), and external carotid artery (ECA) were exposed and the last was dissected distally. ICA was isolated and a 4-0 monofilament nylon with its tip rounded introduced into ECA lumen and then gently advanced into the ICA lumen to occlude the MCA. After 2 h of MCAO, suture was withdrawn to restore blood flow (reperfusion). The incision was sutured and the rats recovered under a heating lamp to maintain rectal temperature at 37 °C ± 0.5 °C. By using this standardized procedure, we obtained large and reproducible infarcted regions involving the temporoparietal cortex and the laterocaudal part of the caudate putamen in ischemic animals. For LXA4 ME treatment, we chose an optimal dose according to our preliminary studies and previous report (Svensson et al., 2007) 0.3 nM LXA4 ME dissolved in 5 µl normal saline, was administered by intracerebroventricular injection immediately after MCA occlusion. In our pilot experiments the difference between I/R group and vehicle-treated rats (5 μl normal saline) was not significant. I/R group without vehicle treatment was used in this study.

4.3.

Assessment of neurological deficits

Neurological deficit in the rats (n = 12 per group) was examined by an observer blinded to the groups at 24 h after reperfusion. The scoring is based on a modified five-point scale as previous reported: grade 0, no observable neurologic deficit (normal); grade 1, flexion of the contralateral torso and the forelimb upon lifting of the animal by its tail (mild); grade 2, circling to the contralateral side but normal posture at rest (moderate); grade 3, leaning to the contralateral side at rest (severe); and grade 4, no spontaneous motor activity (very severe) (Hunter et al., 2000).

Histological examination

At 24 h after reperfusion, rats were deeply anesthetized with chloral hydrate and perfused with heparinized phosphatebuffered saline, followed with 4% paraformaldehyde in phosphate-buffered saline. Tissues were sectioned at a thickness of 5 µm according to the standard procedure. The sections were deparaffinized and hydrated gradually, examined by hematoxylin and eosin, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, respectively.

4.5.1.

TUNEL assay

Apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL) assay in brain at 24 h of reperfusion. The sections (n = 6 for each group) were treated according to the instruction of cell death detection kit (Roche, Basel, Switzerland). TUNEL-positive cells displayed a brown staining within the nucleus in the apoptotic cells. To quantify the DNAfragmented cells after ischemia, the TUNEL-positive cells and total cells in the ischemic cortex at 400× magnification were manually counted. The number of TUNEL-positive cells was expressed as the percentage of total counted cells.

4.5.2.

Immunohistochemistry

Paraffin sections (n = 6 for each group) were immunostained with antibodies against GFAP and OX42 to show astrocyte and microglia as described earlier (Braun et al., 1997). Briefly, after incubation in 0.3% hydroperoxide for 30 min, tissue sections were soaked in 0.01 M citrate buffer (PH 6.0), and heated to the boiling point, blocked by 10% normal goat serum for 30 min, then incubated overnight at 4 °C with chicken-anti-rat GFAP antibody (1:600, Abcam, USA) and mouse-anti-rat OX42 antibody (1:200, Abcam, USA). Following washing in PBS (3 × 5 min), sections were incubated with appropriate secondary biotinylated antibody and then with SABC, developed with diaminobenzidne (DAB), and counterstained with hematoxylin. Parallel brain sections incubated in the absence of

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primary/secondary antibodies served as controls. The sections were visualized with a microscope. The number of GFAPpositive cells and the OX42-positive cells in the ischemic cortex were manually counted in a high-powered field (400×), randomly in non-overlapping five horizons.

4.6.

actin antibody (1:600 dilution, Sigma, USA) antibody according to the manufacturer's instructions and visualized with an enhanced chemiluminescence system (ECL kit; Pierce Biotechnology). The expression in each sample was analyzed with Image J software and quantified as a relative increase over the controls after normalization with β-actin.

Sample processing 4.8.

Rats were deeply anesthetized and killed. Brains were quickly removed to collect the ischemic cortex for all biochemical assays. A 6-mm right coronal section was taken from the area perfused by the MCA, starting 3 mm from the frontal pole (Ashwal et al., 1998). The fresh cortical tissue was collected and frozen immediately in liquid nitrogen and stored at −70 °C until further processing.

Electrophoretic mobility shift assay (EMSA)

Myeloperoxidase (MPO) is an active enzyme correlated with neutrophil accumulation (Matsuo et al., 1994; Shang et al., 2009). After I/R, MPO levels in ischemic cortex were measured with a Rat MPO assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. Samples were measured on a spectrophotometry at 460 nm absorbance. One unit of MPO activity is defined as that which degrades 1 mmol of peroxidase per minute at 25 °C and tissue MPO activities were calculated by using human MPO as a standard.

Nuclear extracts of the ischemic cortex was prepared by hypotonic lysis followed by high salt extraction. EMSA was performed using a commercial kit (LightShift Chemiluminescent EMSA Kit; Pierce; Rockford; USA ). The NF-κΒ oligonucleotide probe sequence was 5′-AGTTGAGGGGAC TTTCCCAGGC3′. Protein–DNA binding assays were performed with 20 µg of nuclear protein. The binding medium contained 5% glycerol, 1% NP40, 1 mM MgCl2, 50 mM NaCl, 0.5 mM EDTA, 2 mM DTT, and 10 mM Tris/HCl, pH 7.5. In each reaction, 20 000 cpm of a radiolabeled probe was included. Sample were incubated at room temperature for 20 min, and the nuclear protein with 32 P-labeled oligonucleotide complex was separated from free 32 P-labeled oligonucleotide by electrophoresis through a 5% native polyacrylamide gel in a running buffer containing 50 mM Tris, pH 8.0, 0.45 mM borate and o.5 mM EDTA. After separation was achieved, the gel was vacuum dried for autoradiography and exposed to Kodak-ray film for 1–2 days at −80 °C.

4.6.2.

4.9.

4.6.1.

Myeloperoxidase Measurement

Malondialdehyde detection

MDA is a biomarker for lipid peroxidation. After I/R, MDA levels in ischemic cortex were measured using MDA and protein assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. Intracellular malondialdehyde concentration was calculated by measuring the maximal absorbance at 532 nm on a spectrophotometer, using N-methy-2-phenylindole as a substrate. All protein concentrations of tissue homogenate samples were determined with BCA method.

4.6.3.

Enzyme-linked immunosorbent assay (ELISA)

At 0 h, 6 h, 12 h, and 24 h of reperfusion, 6 rats in each group were sacrificed and the brain tissue homogenates were obtained from the ischemic cortex. The concentrations of IL1β, TNF-α, IL-10 and TGF-β1 were measured in ischemic brain tissue homogenates using specific ELISA kits according to the manufacturers' instructions (Boster Biological Technology, Wuhan, China).

4.7.

Western blotting

Nuclear protein extracts were prepared according to the instructions provided with the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Pierce Biotechnology, Rockford, USA). In brief, extracts of the ischemic cortex were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the protein bands were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes and immunoblotted with rabbit-anti-rat p65 antibody, for total NF-κΒ detection 1:200 dilution, Boster Biological Technology, Wuhan, China) and mouse-anti-rat β-

Statistical analysis

All data are expressed as mean ± SEM. Differences between the groups were compared using the one-way analysis of variance (ANOVA) with Dunnett's correction. p values less than 0.05 were considered statistically significant.

Acknowledgments This work was supported by the Grant from National Natural Science Foundation of China (no. 30700784).

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