Orally administrated pterostilbene attenuates acute cerebral ischemia–reperfusion injury in a dose- and time-dependent manner in mice

Pharmacology, Biochemistry and Behavior 135 (2015) 199–209

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Orally administrated pterostilbene attenuates acute cerebral ischemia–reperfusion injury in a dose- and time-dependent manner in mice Yu Zhou, Xue-mei Zhang, Ang Ma, Ya-li Zhang, Yan-yi Chen, Hao Zhou, Wen-jun Li, Xin Jin ⁎ Department of Basic Medical Science, Medical College, Xiamen University, Xiamen 361102, PR China

a r t i c l e

i n f o

Article history: Received 5 November 2014 Received in revised form 9 June 2015 Accepted 13 June 2015 Available online 15 June 2015 Keywords: Pterostilbene Cerebral ischemia/reperfusion Oxidative stress Apoptosis Mice

a b s t r a c t Pterostilbene (3,5-dimethoxy-4-hydroxystilbene) is a component of blueberry. It has been reported that longterm treatment with blueberry has a neuroprotective effect. However, it has not been reported whether pterostilbene is effective in attenuating cerebral ischemia/reperfusion (I/R) injury. In the present study, focal cerebral ischemia was induced by middle cerebral artery occlusion for 90 min followed by reperfusion. To observe the dose-dependent effect, pterostilbene (2.5–80 mg/kg, ig) was administered for 3 days before ischemia. To determine the time-dependent effect, pterostilbene (10 mg/kg, ig) was administered as a single dose at 0, 1, or 3 h after reperfusion. Twenty-four hours after I/R, pterostilbene dose-dependently improved neurological function, reduced brain infarct volume, and alleviated brain edema. The most effective dose was 10 mg/kg; the therapeutic time window was within 1 h after I/R and treatment immediately after reperfusion showed the best protective effect. The protective effect is further confirmed by the results that post-ischemic treatment with pterostilbene (10 mg/kg) significantly improved motor function, alleviated blood brain barrier disruption, increased neurons survival and reduced cell apoptosis in cortical penumbra after cerebral I/R. We also found that pterostilbene (10 mg/kg) significantly reversed the increased content of malondialdehyde and the decreased activity of superoxide dismutase in the ipsilateral hemisphere. Furthermore, pterostilbene decreased the oxidative stress markers 4-hydroxynonenal and 8-hydroxyguanosine positive cells in the cortical penumbra. All these findings indicate that pterostilbene dose- and time-dependently exerts a neuroprotective effect against acute cerebral I/R injury. This neuroprotective effect of pterostilbene may be associated with its inhibition of oxidative stress and subsequent neuronal apoptosis in the cortical penumbra. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Stroke is a leading cause of death and disability in many countries (Donnan et al., 2008). Acute ischemic stroke occurs due to the obstruction of blood flow to brain tissue by a thrombus or an embolus, and is characterized by brain infarct and neurological deficits, especially motor dysfunction. Restoration of blood flow to ischemic tissue is the most effective treatment for ischemic stroke. However, the over generation of reactive oxygen species (ROS) during reperfusion attacks protein, lipid and DNA, and then induces apoptosis and necrosis in brain tissue especially in the penumbra area, which plays an important role in determining the final size of the brain infarct (Cherubini et al., 2005). Oxidative stress also leads to blood brain barrier (BBB) disruption after ischemic stroke. The BBB disruption allows normally excluded intravascular proteins and fluid to penetrate into the cerebral parenchymal extracellular space and leads to vasogenic cerebral edema that exacerbates behavioral disability (del Zoppo, 2006; Sandoval and Witt, ⁎ Corresponding author. E-mail address: [email protected] (X. Jin).

http://dx.doi.org/10.1016/j.pbb.2015.06.009 0091-3057/© 2015 Elsevier Inc. All rights reserved.

2008). Considering the important role of ROS in the pathology of stroke, antioxidant therapy for ischemia/reperfusion (I/R)-induced damage attracts intense interest. Previous research indicates that consumption of antioxidant-rich diets have been associated with a decreased risk of stroke in humans (Joshipura et al., 1999). Many animal studies also show that antioxidants protect the brain against cerebral I/R injury (Gao et al., 2006; Karalis et al., 2011; Rodrigo et al., 2013). Pterostilbene (3,5-dimethoxy-4-hydroxystilbene) is a natural stilbene initially isolated from sandalwood and also a component of blueberry (Adrian et al., 2000; Rimando et al., 2004). Many studies have shown that pterostilbene exhibits a variety of biological and pharmacological activities, including anti-inflammatory, anticancer and analgesic activities (Remsberg et al., 2008; McCormack and McFadden, 2013). As a structural analogue of resveratrol, pterostilbene also possesses significant antioxidant activity. It has been reported that pterostilbene exhibited strong anti-oxidant activity against various free radicals such as DPPH, hydroxyl, superoxide and hydrogen peroxide in a concentration dependent manner in vitro (Acharya and Ghaskadbi, 2013; Saw et al., 2014). Previous studies indicated that chronic treatment with blueberry enriched diets protects hippocampal neurons against ischemic injury in

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rats (Sweeney et al., 2002), reduced infarct volume in the cerebral cortex and increased post-stroke locomotor activity (Wang et al., 2005). Recent studies showed that pterostilbene has neuroprotective and cognitive enhancing properties, and in fact improved cognitive performance in a dose-dependent manner in aged rats (Joseph et al., 2008). Dietary supplementation of pterostilbene for 8 weeks significantly improved radial arm water maze function and up-regulated the expression of antioxidative enzymes in aged mice (Chang et al., 2011). However, it still remains unclear whether pterostilbene has a neuroprotective effect on cerebral I/R injury. If it does, the optimal dose and timing of pterostilbene treatment need to be elucidated. Moreover, whether or not its antioxidative effect is involved in this process remains to be clarified. Therefore, in the present study, we first observe the preventive effect of pterostilbene and get a reference dose to further observe its therapeutic effect on acute focal cerebral I/R injury in a mouse model; then we fully evaluate the therapeutic effect of pterostilbene on cerebral I/R injury, focusing on recovery of neurological function, histological outcome, and BBB disruption. Furthermore, we studied the effect of pterostilbene on oxidative stress and neuronal apoptosis in brain tissue after cerebral I/R. 2. Materials and methods 2.1. Chemicals Pterostilbene was purchased from Great Forest Biomedical LTD. (Hangzhou, China) and its purity was 99.2%; 2,3,5-triphenyltetrazolium chloride (TTC), Evans blue (EB), toluidine blue, Tween-80 and trichloroacetic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Experimental animals Experiments were performed in a total of 255 male Kunming mice weighing 25–30 g (Certificate No. SYXK 2008-0003, Experimental Animal Center, Xiamen University, Xiamen, China). Mice were housed under a controlled temperature (22 ± 1 °C) with a 12-h light/dark cycle, and allowed free access to food and water. Experimental protocols were approved by the Animal Care and Use Committee of Xiamen University in compliance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985).

group, vehicle-treated I/R group, and pterostilbene-treated I/R groups (2.5, 10, 40, or 80 mg/kg). Vehicle or pterostilbene was orally administered once a day in a volume of 10 ml/kg for 3 consecutive days before ischemia. Twenty- four hours after I/R, the mice were assessed for neurological function and then sacrificed to collect brain sections for TTC staining (n = 6 mice in sham group and n = 10 mice in the remaining groups). To determine the time-dependent effect of pterostilbene, mice were randomly assigned to five groups: sham group, vehicle-treated I/R group, pterostilbene-treated I/R groups at different time points (0 h, 1 h, or 3 h after reperfusion). Vehicle or 10 mg/kg of pterostilbene (the most effective dose of pterostilbene as pre-ischemia treatment against cerebral I/R injury) was orally administered as a single dose at different time points after reperfusion. Twenty-four hours after I/R, the mice were assessed for neurological function and then sacrificed to collect brain sections for TTC staining (n = 6 mice in sham group and n = 10 mice in the remaining groups). To determine the effect of post-ischemic treatment with pterostilbene on acute cerebral I/R injury, mice were randomly assigned to five groups: sham group treated with vehicle, sham group treated with 10 mg/kg pterostilbene, vehicle-treated I/R group, pterostilbene-treated I/R groups (2.5, 10 mg/kg). For sham group, vehicle or pterostilbene was orally administered at 0 and 2 h after operation; for I/R group, vehicle or pterostilbene was orally administered at 0 and 2 h after reperfusion. In one series, mice were assessed for neurological function and then sacrificed to collect brain tissue for malondialdehyde (MDA) and superoxide dismutase (SOD) activity assays (n = 6 mice in sham group and n = 10 mice in the remaining groups) 24 h after I/R; in the second series, mice were sacrificed to collect brain sections for TTC staining (n = 6 mice in sham group and n = 10 mice in the remaining groups) 24 h after I/R; in the third series, mice were sacrificed for BBB observation 6 h after I/R (n = 6 mice in sham group and n = 10 mice in the remaining groups). To evaluate the effect of post-ischemic treatment with pterostilbene on histological changes, mice were randomly assigned to three groups: sham group, vehicle-treated I/R group, pterostilbene (10 mg/kg)treated I/R group. Vehicle or pterostilbene (10 mg/kg) was orally administered at 0 and 2 h after reperfusion (n = 3 mice in sham group and n = 6 mice in the remaining groups). Twenty-four hours after I/R, mice were sacrificed to collect brain sections for toluidine blue staining, terminal deoxynucleotidyl transferase-mediated dUTP end-labeling (TUNEL) assay, and immunohistochemical analysis. 2.5. Behavior tests

2.3. Induction of focal cerebral I/R injury Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) as we previously reported (Zhou et al., 2012). Mice were anesthetized with chloral hydrate (400 mg/kg, ip.). Briefly, a 6-0 nylon monofilament suture, blunted at the tip and coated with 1% poly-L-lysine, was inserted through the right common carotid artery (CCA) into the internal carotid artery (ICA) and advanced approximately 10 mm distal to the carotid bifurcation to occlude the origin of the middle cerebral artery (MCA) at the junction of the Circle of Willis. Ninety minutes after occlusion, the suture was withdrawn to allow reperfusion. Sham-operated mice underwent identical surgery, except that the intraluminal filament was not inserted. After surgery, mice were kept in a warm box heated by lamps until they woke up and then returned to their home cages. 2.4. Animal experimental protocols and drug application Pterostilbene was dissolved in Tween-80, and then diluted to 10% solution with normal saline. 10% Tween-80 saline solution was used as a vehicle control. To observe the dose-dependent effect of pterostilbene, mice were randomly divided into six groups: sham

The experiment was performed between 9 A.M. and 4 P.M. at standard laboratory conditions (at temperature 25 ± 2 °C and relative humidity 45–50%). To observe the dose- and time-dependent effects of pterostilbene, only the Bederson score was assayed at 24 h after reperfusion. To observed the effect of post-ischemic treatment with pterostilbene, the Bederson score and motor function were assayed at 24 h after cerebral I/R. The order of the assays (Bederson scoring tests, rotarod test, and balance beam walking test) was always the same to maintain identical testing conditions for all animals. All tests were carried out and analyzed by the person blind to the experiment. 2.5.1. Bederson scoring test Bederson scoring test is well established to evaluate the basic neurological dysfunction after ischemic stroke (Bederson et al., 1986). In our experiment, the Bederson behavioral assessments were performed according to our previous report (Zhou et al., 2012). On the test day, the Bederson score of each mouse was given manually by the person blind to the experiment based on a scale from 0 to 3: 0, no deficit; 1, flexion of contralateral forelimb upon lifting of the whole animal by the tail; 2, decrease of thrust toward the contralateral plane; 3, circling to the contra lateral side.

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2.5.2. Accelerated rotarod test Accelerated rotarod test was used to assess motor coordination and balance alterations of mice (Ferrara et al., 2009). The apparatus (YLS-4C, Yiyan Science and Technology Development Co., Ltd. Jinan, Shangdong, China) consisted of a black striated rod (diameter: 3 cm) separated into 5 compartments (width: 6 cm). Before surgery all animals were pretrained for 3 consecutive days (3 trails a day). Firstly, mice were placed on the rod without rotation for 30 s and thereafter, for 120 s, with a low speed rotation from 5 to 40 rpm. Each mouse which fell before 60 s was returned on the rod until mouse was able to stay on it for at least 60 s. After training for 3 days, all animals were able to stay on the accelerated rotarod for at least 1 min. There were no significant differences in the fall latency among all groups before surgery. On the test day, each mouse was placed on the rod rotating from 5 to 40 rpm over 2 min. At each trial, the latency to fall was recorded with a maximum time of 120 s. Passive rotation, accompanying the rod without walking, was considered as a fall. Each mouse was tested 3 times a day with a 10 min inter-trial interval (ITI). The mean of three trials was calculated for each mouse. 2.5.3. Balance beam walking test Balance beam walking test was used to assess motor function in mice, especially hind limb dysfunction (Southwell et al., 2009). The wooden beam was 1 m in length and either 2.8 cm (wide beam) or 1.2 cm (narrow beam) wide. The beam was 50 cm above a padded surface. Before surgery mice were pre-trained on all beams for 3 consecutive days (3 trails a day) until they could walk through the beams stably. In each trial, mouse was placed at the start of the beam and trained to traverse the beam to an enclosed box at the opposite end. On the test day, the performance of each mouse to traverse the beam was scored as follows: 0, if mouse traverses the beam without any slips of the hind limb; 1, if mouse traverses the beam with more than one foot-slip, but less than 50% foot-slips of the affected hind limb; 2, if mouse traverses the beam with more than 50% foot-slips of the affected hind limb; 3, if mouse traverses the beam reluctantly, but the affected hind limb cannot help to move; 4, if mouse is unable to move, but able to stay on the beam; 5, if mouse is unable to stay on the beam. Three trials were performed per mouse for each beam with a 10 min ITI between beams. The mean score of three trials was calculated for each mouse. 2.6. Assessments of infarct volume and ipsilateral edema After evaluation of motor function, these mice were sacrificed by decapitation after anesthesia; the brains were rapidly removed and cut into 5 coronal sections (2 mm thick). Then, these sections were immersed in 2% TTC in phosphate-buffered saline (PBS) at 37 °C for 20 min in the dark, followed by 4% paraformaldehyde in PBS (pH 7.4) overnight. The infarct area of each TTC-stained section was measured by ImageTool 2.0 software (University of Texas Health Science Center, Texas, TX, USA), and infarct volume was calculated as infarct area × thickness (2 mm). The added infarct volumes from all brain sections gave the total infarct volume. To determine the extent of ipsilateral edema, the percentage increase in ischemic hemisphere volume was calculated as (ipsilateral volume − contralateral volume) / contralateral volume × 100%. 2.7. Evaluation of BBB disruption To evaluate the extent of BBB disruption after cerebral I/R, EB leakage in brain tissue was determined 6 h after reperfusion according to our previous report (Zhou et al., 2012). Briefly, 5% EB in 0.9% saline was intravenously administered (4 ml/kg) 1 h before reperfusion. After transcardial perfusion with 0.9% saline, the brain was removed, cut into five 2-mm-thick coronal sections and photographed. The areas of EB leakage on each brain section were measured using an image analysis system and calculated as EB leakage area × thickness (2 mm). The cumulative EB leakage volume from all brain slices was calculated as

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the total EB leakage volume. In addition, brain tissue in the ischemic hemispheres was collected, weighed and homogenized in 50% trichloroacetic acid solution using a homogenizer (IKA, Staufen, Germany). After centrifugation at 3000 × g for 15 min, the supernatant was spectrophotometrically measured at 620 nm. EB leakage content in brain tissue was calculated as μg/g tissue. 2.8. Histological studies 2.8.1. Brain section preparation Twenty-four hours after reperfusion, mice were transcardially perfused with saline followed by 4% paraformaldehyde. Brains were removed and fixed in the same fixative overnight at 4 °C. Before cryosectioning, tissues were cryoprotected with 30% sucrose in PB for 48 h. Serial 10-μm coronal sections (Bregma 0.5 mm–0.14 mm) were cut and used for toluidine blue staining, TUNEL assay, and immunohistochemical analysis. 2.8.2. Toluidine blue staining The 10-μm sections were stained with 1% toluidine blue in PBS for 20 min. After rinsing with double distilled water, they were dehydrated and mounted with Permount (Boshide Biotechnology Company, Wuhan, China). 2.8.3. TUNEL assay DNA fragmentation was detected using a TUNEL assay kit (Boshide Biotechnology Company, Wuhan, China) according to the manufacturer's instructions. Briefly, brain sections were pre-treated with 3% hydrogen peroxide in PBS before staining at room temperature for 10 min to inhibit the endogenous peroxidase activity, and then were digested by proteinase K (20 μg/ml) at room temperature for another 10 min. After incubation with TUNEL reaction mixture containing DIGdUTP and terminal deoxynucleotide transferase at 37 °C for 2 h, the sections were washed and incubated with anti-DIG-Biotin IgG (1:200, Boshide Biotechnology Company, Wuhan, China) for 2 h and then streptavidin horseradish peroxidase (HRP) solution (1:200, Boshide Biotechnology Company, Wuhan, China) for 2 h at room temperature followed by diaminobenzidine (DAB) as colorimetric substrate. Deionized water was used as negative controls in parallel reactions instead of TUNEL reaction mixture. Each procedure was followed by several rinses in tris buffered saline (TBS). 2.8.4. 4-Hydroxynonenal (4-HNE) and 8-hydroxyguanosine (8-OHdG) immunohistochemistry After incubation with 3% hydrogen peroxide for 20 min to inhibit the endogenous peroxidase activity, and 5% normal rabbit serum for 2 h at room temperature, brain sections were incubated overnight at 4 °C with a goat polyclonal antibody against 4-HNE (1:50; a marker for lipid peroxidation, Millipore Corp, Temecula, CA, USA), or a goat polyclonal antibody against 8-OHdG (1:200; a marker for oxidative DNA damage, Calbiochem, EMD Chemicals Inc, Darmstadt, Germany). Subsequently, brain sections were incubated with biotinylated rabbit antigoat IgG (1:200, Boshide Biotechnology Company, Wuhan, China) for 2 h, and then streptavidin HRP (1:200) for 2 h at room temperature. Finally, the sections were visualized using DAB. Normal goat serum was used instead of primary antibody in control sections. 2.8.5. Imaging and analysis In sections stained with toluidine blue, intact neurons were defined as lightly stained cells with a round nucleus. Intact neurons and DAB positive cells were counted in three randomly chosen and nooverlapping squares of identical size (area of 0.13 mm2) located in the cortical penumbra (the region marked by asterisks showed in Fig. 5Aa). The density of intact neurons and DAB positive cells were expressed as the number of cells per mm2. Samples from sham-operated mice

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were prepared as controls. To avoid bias, analyzers were unaware of the experimental design. 2.9. Measurement of MDA content and SOD activity in brain tissue

(one-way ANOVA, F3, 36 = 3.474, P = 0.0037; post-hoc Tukey's test, q = 3.474, P N 0.05, vs. vehicle treated I/R mice, Fig. 1E). Therefore, the optimal timing of post-ischemic treatment was within 1 h after reperfusion.

Twenty-four hours after reperfusion, brain tissue in ipsilateral hemisphere was separated and homogenized in cold saline for MDA content and SOD activity assays. All procedures were performed according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). In brief, MDA content was measured by determining the absorbance of thiobarbituric acid reactive substances at 532 nm by spectrometry. SOD activity was measured using the xanthine oxidase method. Absorbance was determined at 550 nm by spectrometry. All protein concentrations of brain tissue homogenates were determined using a BCA kit (Applygen Technologies Inc., Beijing, China).

3.2. The effect of post-ischemic treatment with pterostilbene on neurological function after acute cerebral I/R

2.10. Statistical analysis

3.2.1. Bederson scoring test Twenty-four hours after cerebral I/R, vehicle treated cerebral I/R mice showed circular behavior, spontaneous depression, and loss of muscle coordination in comparison to sham mice. Scores of the vehicle-treated I/R mice were significantly higher than those of the vehicle-treated sham mice (one-way ANOVA, F4.37 = 26.25, P b 0.01; posthoc Tukey's test, q = 11.06, P b 0.01, Fig. 2A). Pterostilbene (10 mg/kg) significantly decreased the Bederson scores compared to vehicletreated I/R mice (post-hoc Tukey's test, q = 5.16, P b 0.05, Fig. 2A). There was almost no neurological dysfunction in both vehicle- and pterostilbene (10 mg/kg)-treated sham mice (Fig. 2A).

Values are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by post-hoc Tukey's multiple comparison test using Prism 5 for Windows software 2007 (GraphPad Software Inc., San Diego, CA, USA). Values of p b 0.05 were considered statistically significant. 3. Results 3.1. The dose- and time-dependent effects of pterostilbene on acute cerebral I/R injury To evaluate the dose-dependent effect of pterostilbene on cerebral I/ R injury, mice were pre-treated with pterostilbene (2.5–80 mg/kg) for 3 days before inducing cerebral I/R. Our results indicated that pterostilbene 10 mg/kg and 40 mg/kg significantly attenuated neurological dysfunction (one-way ANOVA, F4, 45 = 5.414, P = 0.0014; post-hoc Tukey's test, q = 5.394, P b 0.01 and q = 4.495, P b 0.05, vs. vehicle treated I/R mice, for pterostilbene 10 mg/kg and 40 mg/kg respectively, Fig. 1A), reduced infarct volume (one-way ANOVA, F4, 45 = 5.602, P = 0.0017; post-hoc Tukey's test, q = 5.644, P b 0.01 and q = 4.892, P b 0.01 vs. vehicle treated I/R mice, for pterostilbene 10 mg/kg and 40 mg/kg respectively, Fig. 1B) and edema degree (one-way ANOVA, F4, 45 = 5.498, P = 0.0017; post-hoc Turkey's test, q = 5.997, P b 0.01 and q = 5.130, P b 0.01 vs. vehicle treated I/R mice, for pterostilbene 10 mg/kg and 40 mg/kg respectively, Fig. 1C) compared with vehicle control. In addition, we found that the neuroprotective effect of pterostilbene at 10 mg/kg was superior to that at 40 mg/kg in most indicators especially the infarct volume which is the most important indicator for ischemic stroke treatment. Pre-treatment with pterostilbene at 10 and 40 mg/kg reduced the infarct volume by 71.4% and 61.9% respectively compared with vehicle control (Fig. 1B); and both smaller dose (2.5 mg/kg) and larger dose (80 mg/kg) of pterostilbene had no neuroprotection effects (Tukey's test, q = 1.655, P N 0.05 and q = 2.883, P N 0.05 vs. vehicle treated I/R mice, for pterostilbene 10 mg/kg and 40 mg/kg respectively, Fig. 1B). Therefore, we chose 10 mg/kg of pterostilbene for the further post-ischemic treatment research. To determine the time-dependent effect of pterostilbene on cerebral I/R injury, a single dose of pterostilbene (10 mg/kg, ig) was administered at 0, 1, or 3 h after reperfusion. Pterostilbene (10 mg/kg, ig) administered immediately after reperfusion (0 h) elicited the best protective effect. However, the protective effect of pterostilbene treatment at 1 h after reperfusion is not complete, i.e. pterostilbene only improved neurological score (one-way ANOVA, F3, 36 = 9.611, P = 0.0003; post-hoc Tukey's test, q = 5.002, P b 0.01, vs. vehicle treated I/R mice, Fig. 1D) and alleviated the brain edema degree (one-way ANOVA, F3, 36 = 11.28, P b 0.0001; post-hoc Tukey's test, q = 5.134, P b 0.01, vs. vehicle treated I/R mice, Fig. 1F), but did not reduce the infarct volume

As we know that the early reperfusion is very important for ischemia treatment, and pharmacokinetic studies have shown the half life of oral administrated pterostilbene is about 2 h (Kapetanovic et al., 2011; Azzolini et al., 2014), so to further evaluate the effect of post-ischemic treatment with pterostilbene, mice were treated with pterostilbene (2.5, 10 mg/kg) at 0 and 2 h after reperfusion. Twenty-four hours after reperfusion, the neurological function was evaluated.

3.3. Accelerated rotarod test Twenty-four hours after cerebral I/R, vehicle-treated I/R mice showed poor performances in this motor coordination test. Their fall latency decreased by 87% compared to the sham-operated mice (Fig. 2B). Pterostilbene (2.5 and 10 mg/kg) significantly improved the rotarod performance of I/R mice (one-way ANOVA, F4, 37 = 10.68, P = 0.0001; post-hoc Tukey's test, q = 4.721, P b 0.05 and q = 7.863, P b 0.01 vs. vehicle treated I/R mice, at a dose of 2.5 mg/kg and 10 mg/kg respectively, Fig. 2B). The fall latency of pterostilbene (10 mg/kg)-treated I/R mice was similar to that of the vehicle-treated sham mice (103.6 ± 5.67 s vs. 101.6 ± 12.57 s, Tukey's test, q = 0.1450, P N 0.05, Fig. 2B). There was no difference in fall latency between vehicle-treated and pterostilbene (10 mg/kg)-treated sham mice (Tukey's test, q = 0.076, P N 0.05, Fig. 2B). 3.4. Balance beam walking test Twenty-four hours after cerebral I/R, when vehicle-treated I/R mice passed across the beam, their paralyzed side of the hind limbs could not help their movement forward, and some vehicle-treated I/R mice even could not traverse the beam. Scores of the vehicle-treated I/R mice were significantly higher than those of the sham-operated mice (oneway ANOVA, F4.37 = 6.49, P = 0.0018, post-hoc Tukey's test, q = 5.083, P b 0.05, Fig. 2C). Scores of the pterostilbene (10 mg/kg)treated mice were much lower than those of the vehicle-treated mice (0.67 ± 0.2 vs. 3.5 ± 0.72 points, Tukey's test, q = 5.57, P b 0.01, Fig. 2C). As a control, all sham-operated mice exhibited a successful performance in this test; and there was no difference between vehicletreated and pterostilbene (10 mg/kg)-treated sham mice (Tukey's test, q = 0.463, P N 0.05, Fig. 2C). The same result was attained in the narrow balance beam walking test (Fig. 2D). 3.5. The effect of post-ischemic treatment with pterostilbene on brain infarction and ipsilateral edema after acute cerebral I/R TTC staining of brain sections revealed that brain infarction was mainly located in the MCA territory, such as parietal cortex and striatum

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Fig. 1. The dose- and time-dependent effects of pterostilbene on acute cerebral I/R injury in mice. The neurological deficit score (A, D), infarct volume (B, E) and edema degree (C, F) were determined 24 h after cerebral I/R in mice. To evaluate the dose-dependent effect of pre-ischemic treatment, pterostilbene (2.5–80 mg/kg, ig) was administered for 3 days before inducing cerebral I/R (A–C). To determine the time-dependent effect of post-ischemic treatment, pterostilbene (10 mg/kg, ig) was administrated at the indicated time points (D–F). Data are expressed as mean ± SEM (n = 10 mice in each I/R group). *P b 0.05 and **P b 0.01 vs. vehicle-treated I/R group (one-way ANOVA with Tukey's post hoc test).

(Fig. 3A). Pterostilbene (10 mg/kg) significantly reduced the infarct volume by 79% compared to vehicle control (one-way ANOVA, F2, 27 = 11.13, P = 0.0005, post-hoc Tukey's test, q = 6.281, P b 0.01 Fig. 3C). Especially pterostilbene markedly decreased infarct area of brain sections which include sensorimotor cortex (Fig. 3A, B). The volume of the ipsilateral hemisphere increased by 18.96% compared with that of the contralateral hemisphere, indicating brain edema. Pterostilbene (10 mg/kg) reduced the degree of ipsilateral edema by 79.2% compared to vehicle control (one-way ANOVA, F2, 27 = 8.29, P = 0.0021, post-hoc Tukey's test, q = 5.752, P b 0.01, Fig. 3D).

3.6. The effect of post-ischemic treatment with pterostilbene on BBB disruption after acute cerebral I/R Six hours after reperfusion, remarkable EB extravasations into the cerebral parenchyma were detected in the ipsilateral hemisphere, indicating severe disruption of BBB after cerebral I/R (Fig. 4A). Pterostilbene (10 mg/kg) significantly reduced EB leakage volume (one-way ANOVA, F2, 27 = 10.27, P = 0.0007, post-hoc Tukey's test, q = 6.402, P b 0.05 vs. vehicle treated I/R mice, Fig. 4B) and EB content (one-way ANOVA, F2, 27 = 5.965, P = 0.0089; post-hoc Tukey's test, q = 4.87, P b 0.05,

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Fig. 2. Effect of post-ischemic treatment with pterostilbene on Bederson scoring (A), accelerated rotarod (B), wide balance beam (28 mm) walking (C), and narrow balance beam (12 mm) walking (D) tests 24 h after cerebral I/R in mice. Data are expressed as mean ± SEM (n = 6 mice in vehicle- and pterostilbene (10 mg/kg)-treated sham group and n = 10 mice in the remaining groups). #P b 0.05 and ##P b 0.01 vs. sham; *P b 0.05 and **P b 0.01 vs vehicle-treated I/R mice (one-way ANOVA with Tukey's post hoc test).

Fig. 3. Effect of post-ischemic treatment with pterostilbene on brain infarct volume and edema 24 h after cerebral I/R in mice. (A) Representative brain coronal sections stained with 2% TTC. Typical infarct areas are indicated in white. Bar = 5 mm in A. Infarct volume (B, C) and ipsilateral edema (D) were determined 24 h after cerebral I/R. Data are expressed as mean ± SEM (n = 10 mice in each I/R group). **P b 0.01 vs. vehicle-treated I/R mice (one-way ANOVA with Tukey's post hoc test).

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Fig. 4. Effect of post-ischemic treatment with pterostilbene on BBB disruption 6 h after cerebral I/R in mice. EB leakage volume (A) and EB leakage content (B) were detected 6 h after reperfusion. Data are expressed as mean ± SEM (n = 10 mice in each I/R group). *P b 0.05 vs. vehicle-treated I/R mice (one-way ANOVA with Tukey's post hoc test).

vs. vehicle treated I/R mice, Fig. 4C). No obvious EB leakage was observed in sham operated mice (Fig. 4A). 3.7. The effect of post-ischemic treatment with pterostilbene on neuronal injury in the cortical penumbra after acute cerebral I/R Using toluidine blue staining, we found that intact neuron which was lightly stained and had a round nucleus was almost absent in the cortical penumbra after cerebral I/R. Instead, there were many darkly stained cells with oval or fusiform nuclei in this region (Fig. 5A-b e, B). Compared to vehicle control, pterostilbene increased the number of intact neurons in the corresponding area (Fig. 5A-c f, B). Intact neuron density of the pterostilbene (10 mg/kg)-treated mice was much larger than those of the vehicle-treated I/R mice (548 ± 34.10 cells/mm2 vs. 16.17 ± 6.40 cells/mm2, one-way ANOVA, F2, 12 = 117.2, P b 0.0001; post-hoc Tukey's test, q = 8.817, p b 0.01, vs. vehicle-treated I/R mice, Fig. 5B). As a control, the tissue structure in sham mice was compact, cell outlines were clear and nucleoli were clearly visible. The density of intact neuron in sham group mouse was 1325 ± 99.72 cells/mm2 (Fig. 5A-d, B). For detecting apoptotic cell death, TUNEL staining was performed using an in situ cell death detection kit. Twenty-four hours after cerebral I/R, there were many TUNEL positive cells in the cortical penumbra (Fig. 5A-h). Pterostilbene significantly reduced the number of TUNEL positive cells in the corresponding area (one-way ANOVA, F2, 12 = 30.22, p = 0.0007, post-hoc Tukey's test, q = 6.028, P b 0.05, vs. vehicle-treated I/R mice, Fig. 5A-i, C). Almost no TUNEL positive cells were detected in brain sections of sham mice (Fig. 5A-g). 3.8. The effect of post-ischemic treatment with pterostilbene on oxidative stress in brain tissue after acute cerebral I/R Twenty-four hours after reperfusion, MDA content in ipsilateral hemisphere increased remarkably which indicates lipid peroxidation of brain tissue. Pterostilbene (10 mg/kg) significantly reversed the increased content of MDA (one-way ANOVA, F4, 37 = 16.19, P b 0.0001; post-hoc Tukey's test, q = 4.337, P b 0.05, vs. vehicle-treated I/R mice, Fig. 6A) and the decreased activity of SOD in brain tissue (one-way ANOVA, F4, 37 = 6.287, P = 0.0013, post-hoc Tukey's test, q = 4.26, p b 0.05, vs. vehicle-treated I/R mice, Fig. 6B). There was no difference

between vehicle-treated and pterostilbene (10 mg/kg) -treated sham mice in MDA content (Tukey's test, q = 0.3665, p N 0.05, Fig. 6A) and SOD activity (Tukey's test, q = 0.7991, P N 0.05, Fig. 6B). Furthermore, a number of 4-HNE and 8-OHdG positive cells were found in the cortical penumbra of the vehicle-treated ischemic mice (Fig. 6C-b, e). Pterostilbene (10 mg/kg) significantly decreased the number of 4-HNE positive cells by 56.5% (one-way ANOVA, F2, 12 = 33.22, P = 0.0006; post-hoc Tukey's test, q = 4.337, p b 0.01, vs. vehicle-treated I/R mice, Fig. 6C-c, D) and the number of 8-OHdG positive cells by 55.7% (one-way ANOVA, F2, 12 = 268.6, P b 0.0001; posthoc Tukey's test, q = 20.89, p b 0.01, vs. vehicle-treated I/R mice, Fig. 6C-f, E) compared to vehicle-treated I/R mice. There were almost no 4-HNE or 8-OHdG positive cells in brain sections from sham mice (Fig. 6C-a, d). 4. Discussion In the present study, we report for the first time that pterostilbene dose-dependently protects mice against acute cerebral I/R injury. Evidence is that pterostilbene improves neurological deficit, reduces brain infarct volume, and alleviates brain edema and BBB disruption. The most effective dose is 10 mg/kg; the therapeutic time window is within 1 h after I/R and treatment immediately after reperfusion showed the best protective effect. This protective effect may be mediated by the reduction of oxidative stress and subsequent decrease of neuronal apoptosis in the cortical penumbra. The most important finding is that pterostilbene dose-dependently reduces brain injury after I/R. This result is consistent with previous reports that dietary supplementation with blueberries for 4–6 weeks reduces brain injury after ischemic stroke (Sweeney et al., 2002; Wang et al., 2005). Our results further confirm that pterostilbene is one of the activate ingredients of blueberry to exert its neuroprotective effect. We also found that the neuroprotective effect of pterostilbene is dosedependent. The effective doses are ranged at 10 and 40 mg/kg. It has been reported that pretreatment with pterostilbene (10–40 mg/kg) exerts neuroprotective effect against lipopolysaccharides (LPS) induced brain injury (Hou et al., 2014). In addition, we found that the neuroprotective effect of pterostilbene at 10 mg/kg was superior to that at 40 mg/kg in most indicators, especially the infarct volume which is the most important indicator for ischemic stroke treatment; and both

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Fig. 5. Effect of post-ischemic treatment with pterostilbene on neuronal survival and apoptotic death in cortical penumbra 24 h after cerebral I/R in mice. (A) Representative photographs showing the brain sections stained with toluidine blue (A-b, c, d, e, f) and TUNEL (A-g, h, i). Asterisks mark the cortical penumbra region (A-a, b, c). After cerebral I/R, intact neurons which were lightly stained and had a round nucleus were almost absent in the cortical penumbra. Instead, there were many darkly stained cells with oval or fusiform nuclei (A-b e). Compared to vehicle-treated mice, there were more intact neurons in the brain sections from pterostilbene (10 mg/kg)-treated mice (A-c f). Bar = 100 μm in A-b c; Bar = 25 μm in A-d-i. The density of intact neurons is shown in (B) and the density of TUNEL positive cells is shown in (C). Data are expressed as mean ± SEM (n = 3 mice in sham group and n = 6 mice in the remaining groups). ##P b 0.01 vs. sham; *P b 0.05, **P b 0.01 vs. vehicle-treated I/R mice (one-way ANOVA with Tukey's post hoc test).

smaller dose (2.5 mg/kg) and larger dose (80 mg/kg) had no neuroprotective effects. Therefore, with respect to its neuroprotective effect, pterostilbene displayed a bell-shaped dose–response curve with an optimal dose of 10 mg/kg. This atypical dose response curve could also be found in other neuroprotective antioxidants of diverse structures and activities (Beni et al., 2004; Ye et al., 2007). The lack of effect at the larger dose cannot be explained by the neurotoxicity of pterostilbene, because pterostilbene is known as a very safe agent. It has been reported that

oral intake of a high dose of pterostilbene (3000 mg/kg/day) for about 1 month was not associated with significant local or systemic toxicity in animals (Ruiz et al., 2009). Many studies show that antioxidants at high doses may turn into pro-oxidants via interaction with other oxidants or antioxidants (Beni et al., 2004; Bjelakovic et al., 2012). This may partly explain why pterostilbene at larger dose lose its neuroprotective effect. Actually the pro-oxidant properties of pterostilbene may be an element of cancer chemoprevention due to induction of apoptosis

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Fig. 6. Effect of post-ischemic treatment with pterostilbene on oxidative stress in brain tissue 24 h after cerebral I/R in mice. MDA content (A) and SOD activity (B) in ipsilateral hemisphere were assayed 24 h after reperfusion. (C) Representative photographs showing 4-HNE and 8-OHdG immunostainings in the cortical penumbra (D, E). Bar in C = 25 μm. Data are expressed as mean ± SEM (n = 3 mice in sham group and n = 6 mice in the remaining groups). ##P b 0.01 vs. sham; *P b 0.05, **P b 0.01 vs. vehicle-treated I/R mice (one-way ANOVA with Tukey's post hoc test).

in cancer cells (Schneider et al., 2010; Moon et al., 2013). This result suggests that the neuroprotective effect of pterostilbene against cerebral I/R injury is dose-limited, namely it is effective at an appropriate dose range.

Otherwise, our results indicate that the therapeutic time window of pterostilbene is within 1 h after I/R and treatment immediately after reperfusion showed the best protective effect. It was reported that longterm pretreatment with pterostilbene had a neuroprotective effect

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against brain injury (Joseph et al., 2008; Chang et al., 2012; Hou et al., 2014). In the present study, short-term treatment with pterostilbene can also exert the neuroprotective effect. Pharmacokinetic studies have shown that a peak concentration of pterostilbene was detected in the brain tissue at about 2 h after oral administration of a single dose (20 mg/kg) and was cleared from blood with a half life of about 2 h in rats (Kapetanovic et al., 2011; Azzolini et al., 2014). In our study, pterostilbene treatment immediately after reperfusion showed the best protective effect. This result indicates that pterostilbene mainly exerts its neuroprotective role in the early stage of reperfusion after brain ischemia. This result is in agreement with the notion that early reperfusion is critical for reperfusion treatment (Dyker and Lees, 1998; Olmez and Ozyurt, 2009). Another interesting finding of our study is that post-ischemic pterostilbene dose-dependently decreases the neurological deficit scores and improves motor performances in accelerated rotarod and balance beam walking tests. It is also reported that pretreatment with blueberry diets remarkably improves motor performance in old aged animals (Joseph et al., 2003, 2008; Shukitt-Hale et al., 2007). This result has significant clinical implications because neurological dysfunction, especially motor dysfunction, is the most negative effect of ischemic stroke and the primary cause of adult disability in many countries. This effect may be attributed to the protection of brain tissue especially neurons in sesorimotor cortex by pterostilbene. As we know, the sensorimotor cortex is an important component involved in motor function and the damage to this cortex may lead to behavioral impairments, notably affecting motor function (Mimura et al., 2005; Bu et al., 2007; Sun et al., 2011). Accordingly, we found that pterostilbene (10 mg/kg) markedly decreased infarct area of brain sections which include sensorimotor cortex. It is worth mentioning that during the accelerated rotarod test, which requires better motor coordination and duration, even 2.5 mg/kg pterostilbene shows obvious neurological functional improvement. Fall latency in pterostilbene (10 mg/kg)-treated mice is even similar to that in sham-operated mice. However 2.5 mg/kg pterostilbene did not reduce infarct volume and brain edema. In addition, although pterostilbene is able to pass through BBB, our results showed that short-term treatment with pterostilbene did not affect motor function in sham control mice (Fig. 2). This result is in accordance with others reports that oral pterostilbene (1–40 mg/kg) 1 h before behavior test did not increase the locomotor activity in normal mice (Al Rahim et al., 2013; Hou et al., 2014). Thus it seems that pterostilbene mainly persist motor function after brain injury and the mechanism underlying needs further investigation. Our study further indicates that pterostilbene can attenuate oxidative damage and apoptotic cell death in the ischemic penumbra. We found that pterostilbene decreases both 4-HNE and 8-OHdG positive cells in the ischemic penumbra. 4-HNE is the major oxidation product of polyunsaturated fatty acid and is regarded as the marker of lipid peroxidation (Yoshino et al., 1997; Hayashi et al., 2005). 8-OHdG, on the other hand, is produced when nucleic acid is exposed for oxidative stress, and is also a DNA oxidative stress marker (Hayashi et al., 1999; Itoh et al., 2010). Moreover, by TUNEL assay, we observe that pterostilbene reduces DNA fragmentation, which is a terminal stage of apoptotic DNA degradation in the ischemic penumbra 24 h after I/R. Oxidative stress and apoptotic cell death, especially at the infarct boundary, play important roles in the development of infarct volume (Briyal et al., 2014). Accordingly, we found that pterostilbene reduced infarct volume. All these results indicate that pterostilbene exerts its neuroprotective effect mainly by reducing oxidative stress and DNA fragmentation in the ischemic penumbra. In the early phase of cerebral I/R, because of its high lipophilicity, pterostilbene easily penetrates the cell membranes, nucleus and mitochondria (Chang et al., 2012) and directly scavenge free radicals in the cell. In addition, the antioxidant effect of pterostilbene may also be attributed to its upregulation of anti-oxidant enzymes such as SOD and CAT. In the present study, pterostilbene rescues the SOD activity in brain tissue after cerebral I/R. Pterostilbene could also increase the expression of

SOD and CAT (Bhakkiyalakshmi et al., 2014; Sato et al., 2014). Moreover, pterostilbene is recently reported to increase the expression of two important nuclear factors peroxisome proliferator-activated receptor α (PPARα) (Chang et al., 2011) and nuclear factor-E2 related factor 2 (Nrf2) (Saw et al., 2014), which play important roles in the upregulation of many antioxidant enzymes. Taken together, our study reveals, for the first time, that pre- and post-ischemic treatment with oral pterostilbene are beneficial in improving motor function, reducing infarct volume and attenuating BBB disruption after cerebral I/R in a dose- and time-dependent manner. The most effective dose is 10 mg/kg, and the therapeutic time window is within 1 h after I/R. The suppression of neuronal oxidation stress and apoptosis in the cortical penumbra may be, at least partly, associated with this protective effect. 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