Delayed administration of the nucleic acid analog 2Cl-C.OXT-A attenuates brain damage and enhances functional recovery after ischemic stroke

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Research Report

Delayed administration of the nucleic acid analog 2Cl-C.OXT-A attenuates brain damage and enhances functional recovery after ischemic stroke Naohiko Okabea, Emi Nakamuraa, Naoyuki Himia, Kazuhiko Naritaa, Ikuko Tsukamotob, Tokumi Maruyamac, Norikazu Sakakibarac, Takehiro Nakamurad, Toshifumi Itanod, Osamu Miyamotoa,n a

Department of Physiology 2, Kawasaki Medical University, 577 Matsushima, Kurashiki, Okayama, Japan Department of Pharmaco-Bio-Informatics, Faculty of Medicine, Kagawa University, Ikenobe, Miki, Kagawa, Japan c Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Shido, Sanuki, Kagawa, Japan d Department of Neurobiology, Faculty of Medicine, Kagawa University, Ikenobe, Miki, Kagawa, Japan b

art i cle i nfo

ab st rac t

Article history:

2Cl-C.OXT-A (COA-Cl) is a novel nucleic acid analog that enhances angiogenesis through

Accepted 6 February 2013

extracellular signal-regulated kinase 1 or 2 (ERK1/2) activation. ERK1/2 is a well-known kinase

Available online 14 February 2013

that regulates cell survival, proliferation and differentiation in the central nervous system.

Keywords:

We performed in vitro and in vivo experiments to investigate whether COA-Cl can attenuate

COA-Cl

neuronal damage and enhance recovery after brain ischemia. In primary cortical neuron

Brain ischemia

cultures, COA-Cl prevented neuronal injury after 2 h of oxygen-glucose deprivation. COA-Cl

Purinergic receptor

increased phospho-ERK levels in a dose-dependent manner and COA-Cl-induced neuroprotec-

ERK 1/2

tion and ERK1/2 activation was inhibited by suramin or PD98059. The effect of COA-Cl was

Angiogenesis

evaluated in vivo with 60 min of middle cerebral artery occlusion combined with bilateral

Synaptogenesis

common carotid artery occlusion. COA-Cl or saline was injected intracerebroventricularly 5 min after reperfusion. COA-Cl significantly reduced infarct volume and improved neurological deficits upon injection of 15 or 30 mg/kg COA-Cl. Moreover, COA-Cl reduced the number of TUNEL positive cells in ischemic boundary, while rCBF was not significantly changed by COA-Cl administration. We also evaluated the effect of delayed COA-Cl administration on recovery from brain ischemia by continuous administration of COA-Cl from 1 to 8 days after reperfusion. Delayed continuous COA-Cl administration also reduced infarct volume. Furthermore, COA-Cl enhanced peri-infarct angiogenesis and synaptogenesis, resulting in improved motor function recovery. Our findings demonstrate that COA-Cl exerts both neuroprotective and neurorestorative effects over a broad therapeutic time window, suggesting COA-Cl might be a novel and potent therapeutic agent for ischemic stroke. & 2013 Elsevier B.V. All rights reserved.

Abbreviations: COA-Cl,

2Cl-C.OXT-A; ERK,

activated protein kinase; MCAO,

extracellular signal-regulated kinase; LDH,

middle cerebral artery occlusion; MEK,

lactate dehydrogenase; MAPK,

MAP kinase kinase; OGD,

rCBF, regional cerebral blood flow; TUNEL, terminal deoxynucleotidyl transferase-dUTP nick end labeling n Corresponding author. Fax: þ81 86 462 1199. E-mail address: [email protected] (O. Miyamoto). 0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.02.009

mitogen-

oxygen-glucose deprivation;

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Fig. 1 – COA-Cl protects neurons from OGD-induced neuronal injury in an ERK1/2 activation-dependent manner: (A) concentration response of COA-Cl-induced neuroprotection. After 2 h of OGD, primary cortical neurons were incubated with COA-Cl for 24 h. Po0.01 vs. vehicle-treated OGD control. Data are normalized to the amount of LDH released from vehicle-treated cells after OGD (100%) and are corrected for baseline LDH release (0%) measured in control cell cultures for each experiment. (B) Concentration response of COA-Cl-induced ERK1/2 activation. After 2 h of OGD, primary cortical neurons were treated with COA-Cl for 15 min. Representative western blots and semi-quantitative data of p-ERK and total ERK1/2 activation are shown. Po0.05, Po0.01 vs. vehicle control. (C) Co-exposure of the neurons to COA-Cl (100 lM) and PD98059 abolished the protective effect of COA-Cl. Po0.05, Po0.001 vs. vehicle-treated OGD control. ]]]Po0.001 vs. COA-Cl without antagonists. (D) PD98059 (10 lM) abolished COA-Cl induced ERK1/2 activation. Po0.01 vs. vehicle control. ]]]Po0.001 vs. COA-Cl without antagonists. (E) Co-exposure of the cells to COA-Cl and suramin abolished the protective effect of COA-Cl. Po0.05 vs. vehicle control; ]Po0.05 vs. COA-Cl without antagonists. (F) Suramin (100 lM) abolished COA-Cl-induced ERK1/2 activation. Po0.001 vs. vehicle control. ]]Po0.01 vs. COA-Cl without antagonists. Data in such graphs show the mean7SD derived from at least 4 independent experiments.

1.

Introduction

Recent investigations into the pathophysiological events that follow acute ischemic stroke suggest an important role for angiogenesis, which results in improved collateral circulation

(Wei et al., 2001; Gu et al., 2001) and may impact medium-tolong term recovery (Krupinski et al., 1994). Several substances that promote angiogenesis, such as fibroblast growth factors, platelet-derived growth factors, and vascular endothelial growth factors, are known. However, all these growth factors

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are proteins with high-molecular weights, ranging from 15,000 to 30,000 Da. Thus, they have problems of low absorbability and stability. 2Cl-C.OXT-A (COA-Cl) is a soluble and stable nucleic acid analog with a molecular weight of 284 Da. We previously reported that COA-Cl shows a strong angiogenic activity in human umbilical vein endothelial cells (HUVECs), chicken chorioallantoic membrane, and rabbit cornea via extracellular signal-regulated kinase 1 or 2 (ERK1/2) activation (Tsukamoto et al., 2010). Although the effect of COA-Cl in the central nervous system is unknown, previous data suggest that COA-Cl might enhance functional recovery after brain ischemia via angiogenesis. We hypothesized that in addition to having angiogenic effects, COA-Cl may modulate neuronal survival after ischemic brain injury, because ERK1/2 is known to regulate cell survival, proliferation, and differentiation in many tissues, including the central nervous system (Xia et al., 1995; Harada et al., 2001; Xiao et al., 2007). In the present study, we investigated the effects of COA-Cl on ischemic neural injury using in vitro and in vivo stroke models.

2.

Results

2.1. COA-Cl protects neurons from oxygen-glucose deprivation (OGD) induced neuronal injury in an ERK1/2 activation dependent manner After 2 h of OGD, primary cortical neuron cultures were incubated with COA-Cl for 24 h. Cell death was evaluated 24 h after OGD by measuring level of LDH released into the media from dead or dying cells. As shown in Fig. 1A, COA-Cl showed significant neuroprotective effects at 100 (Po0.01) and 500 mM (Po0.01). Next, we examined ERK1/2 activation to elucidate whether COA-Cl protects neuron using the same pathway as used in its angiogenic function. Neuron cultures were incubated with COA-Cl for 15 min after OGD, followed by protein extraction and western blot analyses. COA-Cl treatment resulted in a dose-dependent ERK1/2 activation (Fig. 1B, 50 mM; Po0.05, 100 mM and 500 mM, Po0.01). Furthermore, the COA-Cl-induced neuroprotection (Fig. 1C, Po0.001) as well as ERK1/2 activation (Fig. 1D, Po0.001) were abolished by the specific mitogen-activated protein kinase kinase (MEK) inhibitor PD98059, indicating that COA-Cl protects cultured cortical neurons from OGD-induced cell death via an MEK and ERK1/2-mediated mechanism. Although the molecular pathway by which COA-Cl activates mitogen-activated protein kinase (MAPK) has not been reported previously, we hypothesized that the protective effects of COA-Cl might be due to activation of the purinergic receptor-ERK1/2 pathway because COA-Cl is structurally similar to adenosine triphosphate. To test this hypothesis, cell cultures were incubated with COA-Cl and the purinergic receptor antagonist suramin. As expected, suramin abolished the neuroprotective effect of COA-Cl (Fig. 1E, Po0.05) in a dose-dependent manner and inhibited ERK1/2 activation (Fig. 1F, Po0.01). To further clarify the molecular pathways activated by COA-Cl, ERK1/2 phosphorylation was examined using antagonists that interfere with the purinergic receptor-ERK1/2

Fig. 2 – COA-Cl activates ERK1/2 through P2X receptor-, Ca2þ-, and PKC-dependent pathway. After 2 h of OGD, neurons were treated with COA-Cl for 15 min. The purinergic receptors pathway antagonists were added 5 min prior to COA-Cl and incubated together with COA-Cl. Representative western blots and semi-quantitative data of pERK and total ERK1/2 activation are shown: (A) PPADS (100 lM) reduced COA-Cl-induced ERK1/2 activation. Po0.001 vs. vehicle control. ]]]Po0.001 vs. COACl without antagonists. (B) U73122 (5 lM), wortmannin (0.5 lM), or LY294002 (20 lM) did not significantly decrease COA-Cl induced ERK1/2 activation. Po0.05, Po0.01 vs. vehicle control. (C) BAPTA-AM (10 lM) and GF109203X (10 lM) reduced COA-Cl induced ERK1/2 activation. Po0.001 vs. vehicle control. ]]Po0.01, ]]]Po0.001 vs. COA-Cl without antagonists. Data in each graphs show the mean7SD derived at least 4 independent experiments.

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Table 1 – Physiological parameters for the saline and COA-Cl treated groups. Values are shown as the mean7S.D. BT (1C)

BP (mmHg)

HR (/min)

pH

pCO2 (mmHg)

pO2 (mmHg)

Saline (n ¼12) Pre-MCAO MCAO Post-MCAO

37.470.29 37.470.26 37.570.35

94.875.4 99.4716 86.976.9

416727 430730 416736

7.4670.04 7.4470.02 7.4370.03

33.772.42 32.772.1 32.472.2

133711 127712 130711

COA-Cl (n ¼ 12) Pre-MCAO MCAO Post-MCAO

37.470.29 37.370.30 37.470.26

97.675.3 99.0710 91.975.0

421735 438743 428735

7.4770.03 7.4370.04 7.4170.03

32.272.6 33.673.7 33.773.25

130714 128716 128715

Body temperature, BT; mean arterial blood pressure, BP; heart rate, HR; arterial pH, pH; partial pressure of carbon dioxide, pCO2; partial pressure of oxygen, pO2.

pathway at different levels. Purinergic receptors consist of 2 main families: a P2X family of ligand-gated ion channel receptors and a P2Y family of G-protein-coupled receptors. We first evaluated the effect of the selective P2X receptor antagonist PPADS on COA-Cl-induced ERK1/2 activation. Neurons were pretreated with PPADS for 5 min and then incubated with COA-Cl for 15 min. As shown in Fig. 2A, PPADS markedly decreased COA-Cl-induced ERK1/2 activation (Po0.001). In the next experiment, we inhibited downstream targets of P2Y receptors to determine whether COA-Cl activates P2Y receptors. Neurons were treated with the selective phospholipase C inhibitor U73122 or the selective phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002. U73122 did not alter COA-Cl-induced ERK1/2 activation (P40.05). Wortmannin (P40.05) and LY294002 (P40.05) slightly reduced ERK1/2 activation, but the reductions were not statistically significant (Fig. 2B). These results indicate COA-Cl predominantly activates P2X receptors, although P2Y receptors may also be stimulated to some extent. Because P2X receptors regulate intracellular Ca2þ levels through ligand-stimulated increase in calcium permeability and activate ERK1/2 via protein kinase C (PKC), we performed further studies to determine if intracellular Ca2þ and PKC were involved in COA-Cl-induced ERK activation. We treated neurons with the membrane permeable calcium chelator BAPTA-AM or the selective PKC inhibitor GF109203X. Both BAPTA-AM (Po0.01) and GF109203X (Po0.001) significantly decreased COA-Cl induced ERK1/2 activation (Fig. 2C).

2.2. The effect of acute COA-Cl treatment after brain ischemia To validate whether COA-Cl protects against neuronal ischemia in vivo, transient brain ischemia was induced by 60 min of MCAO, combined with bilateral common carotid artery occlusion. Saline or COA-Cl (6–30 mg/kg) was administered by intracerebroventricular injection 5 min after the onset of reperfusion. There were no significant differences in any physiological parameters among the groups (Table 1). Though all rats survived until behavioral assessment, 3 rats were excluded because of insufficient neurological deficits according to our clinical score criteria (COA-Cl 6 mg/kg; 1 rat, COACl 15 mg/kg; 2 rats). The corrected infarct volume (infarct

volume—edema) in the saline-treated group was 287.97 72.4 mm3 as determined by TTC staining 24 h after reperfusion. When 6 mg/kg of COA-Cl was injected, no significant effect was observed on either the clinical score or corrected infarct volume. However, when 15 or 30 mg/kg of COA-Cl was injected, the clinical score significantly improved, and corrected infarct volume was reduced (Fig. 3A–C, clinical score; 15 mg/kg: Po0.05, 30 mg/kg: Po0.05, corrected infarct volume; 15 mg/kg: Po0.05, 30 mg/kg: Po0.05). The maximum reduction in corrected infarct volume was achieved when 30 mg/kg of COA-Cl was injected; the corrected infarct volume was reduced to 167.3746.8 mm3, which was 41.8% lesser than that for the saline-treated control. In the separate experiment, the effects of COA-Cl on rCBF and apoptosis were also examined after acute COA-Cl administration to investigate the mechanism of COA-Cl mediated neuroprotection in vivo. Cortical rCBF was continuously monitored using a laser Doppler probe from 10 min prior to brain ischemia to 60 min after the onset of reperfusion. rCBF decreased to 22.974.6% and 22.775.4% of the baseline values after induction of brain ischemia in saline and COACl-treated group, respectively. These decreased rCBF values were sustained during whole periods of brain ischemia. The rCBF increased after reperfusion, and the maximum values were 113.9730.0% and 138.0742.6% in saline and COA-Cltreated group, respectively. Although COA-Cl-treated group showed a tendency for increased values after drug administration, two-way repeated-measures ANOVA did not reveal significant difference between groups (P¼ 0.4017, Fig. 4A). Furthermore, COA-Cl treatment did not affect the other physiological parameters at 1 h after the onset of reperfusion (Table 2). Apoptotic cell death was evaluated 24 h after brain ischemia using TUNEL staining. TUNEL positive cells which exhibited shrunken cell bodies and condensed nuclei were distributed both in cortex and striatum. As shown in Fig. 4B, TUNEL positive cells were sparsely distributed in ischemic boundary zone in COA-Cl-treated rat, whereas they were densely distributed in saline-treated rat. The numbers of TUNEL positive cells in ischemic boundary zone were 632.3767.9/mm3 and 411.5787.7/mm3 in saline and COA-Cltreated group, respectively. Student’s t-test revealed significant reduction of the number of the TUNEL positive cells in COA-Cl-treated group (Fig. 4C, P ¼0.0002).

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Fig. 3 – Acute intracerebroventricular COA-Cl decreases infarct volume and improves neurological function after brain ischemia. COA-Cl (6–30 lg/kg) or saline was administered by intracerebroventricular injection 5 min after the onset of reperfusion. Infarct volumes were evaluated using TTC staining 24 h after reperfusion: (A) representative TTC staining from a saline-treated and 15 lg/kg COA-Cl-treated rat, (B) COA-Cl improved the clinical score 24 h after brain ischemia. n ¼ 6 for each group, Po0.05 vs. saline-treated group. Data show the median and IQR. (C) COA-Cl significantly reduced corrected infarct volume. n ¼ 6 for each group, Po0.05 vs. saline-treated group. Data show the mean7SD.

2.3. Effect of delayed chronic COA-Cl treatment after brain ischemia To evaluate the effect of continuous COA-Cl treatment on recovery after brain ischemia, rats were implanted with an osmotic mini-pump 1 day after induction of brain ischemia/ reperfusion. COA-Cl (1.5 mg/kg/h) or the same volume of saline was administered continuously into the cerebral ventricle for 7 days. BrdU (50 mg/kg) was given by intraperitoneal injection once daily during the same period (Fig. 5A). One rat was excluded before drug administration according to our clinical score criteria. Twenty-four hours after brain ischemia, there were no differences in body weight, clinical score, or the rotarod test results between the 2 groups. COA-Cl slightly improved the clinical score 6 and 13 days after ischemia, but the differences were not statistically significant (Fig. 5B). However, COA-Cl significantly extended the retention time in rotarod tests. Two-way repeated-measures ANOVA revealed a significant effect of COA-Cl (P¼ 0.0035), and post hoc analyses showed a significant difference between COA-Cl and saline treatment on days 3 (Po0.05) and 13 (Po0.001) after brain ischemia (Fig. 5C).

To study the mechanism by which COA-Cl enhances motor function recovery, rats were sacrificed on day 13 for histological examinations. First, brain damage (infarctþ atrophy) and remaining healthy tissue volume were evaluated by HE staining. The brain damage and healthy tissue volume in the saline-treated group were 189.7752.8 mm3 and 303.0751.7 mm3, respectively. COA-Cl administration significantly reduced brain damage and increased healthy tissue volume; these volumes were 127.5742.9 mm3 and 388.9767.8 mm3, respectively (Fig. 6B and C, brain damage; P¼ 0.0491, healthy tissue volume; P¼ 0.0206). These were 32.8% reduction of brain damage and 28.3% increase of remaining healthy tissue despite delaying treatment for 1 day. To verify the effect of the late administration of COA-Cl, we carried out time course study of infarct volume in our MCAO. As shown in Fig. S1, brain edema and corrected infarct volume expanded rapidly in first 24 h. While edema volume decreased following 48 h (Po0.01, 24 h vs. 72 h), corrected infarct volume slowly increased with time (Po0.05, 24 h vs. 72 h). The corrected infarct volume at 72 h after brain ischemia increased by 39.3% compared to that at 24 h after brain ischemia. Healthy tissue volume also gradually decreased with time until 72 h after brain ischemia (Po0.05, 24 h

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Fig. 4 – Acute intracerebroventricular COA-Cl decreases the number of TUNEL positive cells in ischemic boundary zone without significant effect on rCBF after brain ischemia: (A) rCBF was measured for 10 min before brain ischemia to establish the baseline value. Then brain ischemia was induced for 60 min by MCAO combined with bilateral CCA occlusion. Saline or COA-Cl was administrated intracerebroventricularly 5 min after the onset of reperfusion. rCBF decreased to less than 25% of the baseline values after induction of brain ischemia and recovered to more than 100% of the baseline values after the reperfusion. While COA-Cl slightly increased the rCBF during reperfusion, the difference between the groups was not statistically significant (P¼0.4017, n¼8). Data show the mean7SD. For TUNEL assay, rats were subjected to brain ischemia for 1 h and saline or COA-Cl was injected intracerebroventricularly 5 min after the onset of reperfusion. Following 24 h reperfusion, rats were sacrificed and apoptotic cell death was evaluated using TUNEL staining. (B) Representative picture of TUNEL staining. Pictures show the ischemic boundary area in low magnification (upper column,  10 objective, scale bar 400lm) and high magnification (lower column,  40 objective, scale bar 100 lm). More TUNEL positive cells exist in the ischemic boundary zone of saline-treated brain section (left) compared to COACl-treated one (right). (C) COA-Cl treatment significantly reduced the number of TUNEL positive cells. n¼ 7 for each group, Po0.001 vs. saline-treated group. Data show the mean7SD.

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Table 2 – Physiological parameters for the saline and COA-Cl treated groups 1 h after the onset of reperfusion. Values are shown as the mean7S.D.

Saline (n ¼8) COA-Cl (n¼ 8)

BT (1C)

BP (mmHg)

HR (/min)

pH

pCO2 (mmHg)

pO2 (mmHg)

37.370.1 37.470.8

88.8711.7 81.1710.7

389724 384735

7.3570.03 7.3870.03

35.577.5 35.774.4

10179 10879.8

Body temperature, BT; mean arterial blood pressure, BP; heart rate, HR; arterial pH, pH; partial pressure of carbon dioxide, pCO2; partial pressure of oxygen, pO2.

Fig. 5 – Delayed continuous COA-Cl administration improves motor function recovery after brain ischemia. Rats were subjected to brain ischemia and continuously administered saline or COA-Cl (5 lg/kg/h) for 7 days beginning on 1 day after ischemia onset: (A) time course diagram for various experiments in this study and (B) COA-Cl did not significantly improve the clinical score (n ¼ 7 for each group). Data show the median and IQR. (C) COA-Cl significantly improved rotarod test results. (n ¼7 for each group), Po0.05, Po0.001 vs. saline control at the same time point. Data show the mean7SD.

vs. 72 h). These results indicate delayed COA-Cl treatment prevented the prolonged progression of brain destruction. Considering the confounding effect of infarct volume reduction on behavior test result, we compared the results of behavior tests in animals from different treatment groups with that displayed similar infarct sizes (Fig. S2A, brain damage range from 100 to 200 mm3) to address the specific effects of COA-Cl on motor recovery without the confounding effects of this agent on infarct volume. In this range, the infarct volumes were 162.6741.1 mm3 in saline-treated group (n¼ 4) and 141.7728.2 mm3 in the COA-Cl-treated group (P¼ 0.3945; n¼5). While COA-Cl showed almost no effect on clinical score (Fig. S2B), COA-Cl still showed significant improvement in motor recovery (Fig. S2C). Two-way repeated-measures ANOVA revealed a significant effect of COA-Cl (P¼ 0.0138), and post hoc analyses showed a significant difference between COA-Cl and saline treatment on 13 days after brain ischemia (Po0.01). This result indicates that COA-Cl not only decreases the infarct volume and

attenuates motor function disturbances, but also enhances motor function recovery. Next, we evaluated the effect of COA-Cl on neurorestorative responses including angiogenesis, neurogenesis, and synaptogenesis. In this series of experiments, angiogenesis, neurogenesis, and synaptogenesis in animals with similar infarct volumes were also compared to exclude effect of infarct volume (similar to the rotarod tests). Angiogenesis was evaluated using BrdU and laminin double staining. BrdUþ/lamininþ cells were observed in the ischemic border in the groups treated with both saline and COA-Cl (Fig. 7A and B). The numbers of BrdUþ/lamininþ cells in the penumbra region in the saline treated rats was 3.9 fold more than that in normal rats. As expected from a previous report (Tsukamoto et al., 2010), the number of BrdUþ/ lamininþ cells in rats treated with COA-Cl after brain ischemia was 2.1 and 8.5 fold more than that in saline-treated and normal rats, respectively (Fig. 7C, P¼ 0.001). The number of

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Fig. 6 – Delayed continuous COA-Cl administration decreases infarct volume despite delayed treatment. Rats underwent brain ischemia and were continuously treated with saline or COA-Cl from days 1 to 8. Animals were sacrificed on day 13 after brain ischemia/reperfusion. Infarct volume was evaluated using HE staining: (A) representative HE staining images from saline-treated and COA-Cl-treated rat. (B)COA-Cl significantly reduced brain damage. (n¼ 6 for each group). Po0.05 vs. saline treated group. (C) COA-Cl significantly increased remaining healthy tissue volume. (n ¼ 6 for each group). Po0.05 vs. saline treated group. Data in each graphs show the mean7SD.

BrdUþ/lamininþ cells and brain damage showed weak correlation (Fig. S3A, R2 ¼ 0.237, P¼ 0.1085), and enhanced angiogenesis in COA-Cl-treated rats were also demonstrated by analysis with brain damage matched animals (Fig. 7D, P¼ 0.0031). In the neurogenesis study, BrdU and DCX double staining showed increased number of neuroblasts migrating from the subventricular zone to the striatum in both groups (Fig. 8A and B). Although the number of BrdUþ/DCXþ cells was increased 9-fold from normal level in COA-Cl group, it was nearly half of saline group (Fig. 8C, P¼ 0.013). The number of BrdUþ/DCXþ cells and brain damage showed a good correlation (Fig. S3B, R2 ¼ 0.6154, P¼ 0.0025), indicating that the number of BrdUþ/DCXþ cells depends on brain damage rather than COA-Cl. Actually, no significant difference was observed between groups in brain damage matched analysis (Fig. 8D, P¼ 0.4584). The effect of COA-Cl on synaptogenesis was evaluated using an antibody against the presynaptic marker synaptophysin. Synaptophysin expression in the COA-Cl-treated group was significantly more than that in the saline-treated group (Figs. 9A and B, P¼ 0.0006). Synaptophysin expression was weakly correlate with brain damage (Fig. S3C, R2 ¼0.2238, P¼ 0.1204), and brain damage matched analysis also demonstrated significantly increased synaptophysin expression in COA-Cl-treated group (Fig. 9C, P ¼0.0068).

Finally, we analyzed correlation between each histological parameters and behavioral outcome. As shown in Fig. 10, brain damage correlated negatively with the result of rotarod on day 13 after brain ischemia (Fig. 10A, R2 ¼0.2213, P ¼0.1227). Interestingly, the numbers of BrdUþ/lamininþ cells and synaptophysin expression showed more strong positive correlation with behavioral outcome than brain damage (Fig. 10B and D, the numbers of BrdUþ/lamininþ cells; R2 ¼0.4030, P¼ 0.0266, synaptophysin; R2 ¼ 0.4286, P ¼0.0209), whereas the number of BrdUþ/DCXþ cells and the result of rotarod on day 13 after ischemia showed very weak correlation (Fig. 10C, R2 ¼0.01618, P¼ 0.6936).

3.

Discussion

The present study demonstrates that COA-Cl exerts neuroprotective effects in vitro and in vivo. Importantly, COA-Cl was effective when it was administered after ischemia and reperfusion, and protection was observed even when the treatment was delayed for 1 day. These results suggest that COA-Cl has a wide therapeutic time window. In addition to its neuroprotective effects, COA-Cl increased angiogenesis and synaptogenesis after brain ischemia. Comparisons of animals with similar infarct sizes and the correlation analysis between histological parameters and functional outcome

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Fig. 7 – COA-Cl enhances angiogenesis after brain ischemia. BrdU was injected once daily during the same period as COA-Cl treatment, and rats were killed on 13 days after brain ischemia. Brain sections were immunostained for BrdU and laminin: (A) representative fluorescence images of laminin and BrdU double staining in the penumbral region are shown. Scale bar¼ 20 lm. (B) Representative fluorescence images of laminin and BrdU double staining from saline treated group and COA-Cl treated group. Scale bar ¼40 lm. (C) COA-Cl significantly increased the number of BrdUþ/lamininþ cells (n¼ 7 for each group). Po0.01 vs. saline-treated group. (D) Brain damage matched subgroup analysis (saline; n¼ 4, COA-Cl; n ¼ 5) also demonstrated significant increase of the number of BrdUþ/lamininþ cells in COA-Cl treated group. The dotted line in the bar graph shows the basal level of normal rats. Po0.01 vs. saline-treated group. Data in each graphs show the mean7SD.

demonstrated that COA-Cl facilitates neuronal recovery by stimulating angiogenesis and synaptogenesis.

3.1. Neuroprotective effects of COA-Cl and in vitro purinergic receptor activation Because COA-Cl is a novel nucleic acid analog, its mechanism of action is largely unknown. In this study, we demonstrate that similar to the angiogenic effect of COA-Cl in HUVECs

(Tsukamoto et al., 2010), neuroprotective effect in neuronal cultures is dependent on ERK1/2 activation. ERK1/2 is activated by other well-known neuroprotective agents, including estrogen (Singer et al., 1999), erythropoietin (Kilic et al., 2005), brain-derived neurotrophic factor (Hetman et al., 1999) and leptin (Zhang et al., 2007). ERK1/2 has also been reported to promote cell survival by inhibiting pro-apoptotic signal proteins, including caspase-9 (Allan et al., 2003) and Bim-EL (Luciano et al., 2003). This anti-apoptotic effect of ERK1/2 is

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Fig. 8 – COA-Cl does not enhance neurogenesis after brain ischemia: (A) representative fluorescence images of DCX and BrdU double staining are shown. Scale bar ¼20 lm. (B) Representative fluorescence images of DCX and BrdU double staining from saline-treated group and COA-Cl-treated group. Scale bar ¼200 lm. COA-Cl significantly decreased the number of BrdUþ/ DCXþ cells (n ¼7) (C). However, significant difference was not observed when animals with similar infarct volumes were compared (D) (saline; n¼ 4, COA-Cl; n ¼5). The dotted line in the bar graph shows the basal level of normal rats. Data in each graphs show the mean7SD.

consistent with our in vivo results. Our results also elucidated an important role for purinergic receptors, particularly, the P2X receptors, in COA-Cl-induced neuroprotection. Although some reports described a harmful effect of P2X receptor activation on neuronal cell death (Sugiyama et al., 2010; Kong et al., 2005), a recent study showed that the P2  7 receptor agonist, BzATP, prevented cerebellar granule neuron apoptosis by activating PKC- and ERK1/2-dependent

pathways (Ortega et al., 2009, 2011). The conflicting evidence on the effects of P2X receptor activation on neuronal injury might arise from different P2X receptor expression levels, combined with other purinergic receptor activation or receptor selectivity of the ligands that were evaluated. Thus, to clarify the precise mechanism of action, the specificity of COA-Cl to each P2X receptor subtypes should be examined by knock down or knock out experiments in the future study.

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Fig. 9 – COA-Cl increases synaptophysin expression after brain ischemia. Brain sections were stained using the synaptophysin antibody, and the optical density (OD) of synaptophysin immunoreactivity in the penumbral region was evaluated: (A) representative fluorescence images of synaptophysin staining. Scale bar¼ 20 lm. (B) COA-Cl treatment significantly increased the synaptophysin immunoreactivity OD compared to the saline treatment (n ¼7 for each group). Po0.001 vs. saline treated group. (C) Brain damage matched subgroup analysis (saline; n¼ 4, COA-Cl; n¼ 5) also demonstrated significant increase of the synaptophysin immunoreactivity OD in COA-Cl treated group. The dotted line in the bar graph shows the basal level of normal rats. Po0.01 vs. saline treated group. Data in each graphs show the mean7SD.

3.2.

Neuroprotective effect of COA-Cl in vivo

While many factors, including the administration route, dose, and time of administration, affect drug efficacy, the therapeutic time window (Baron et al., 1995) is especially important for the treatment of ischemic brain injury. In the present study, COA-Cl showed significant neuroprotection even if injected a day after the onset of reperfusion. Although neuroprotection following delayed treatment is unusual, effective delayed therapy has been recently reported for some drugs. For example, intravenous injection of recombinant human erythropoietin at 6, 24 and 48 h after embolic MCAO showed a 28% reduction in infarct volume (Wang et al., 2007), and vascular endothelial growth factor reduced infarct volume by 35% when it was continuously administered by intracerebroventricular injection beginning 24 h after MCAO performed using the suture method (Sun et al., 2003). In this study, COA-Cl reduced infarct volume by 32.8%, and its therapeutic effect and time window was comparable to that

reported for the drugs used in other studies. However, COA-Cl is a small molecule with excellent biostability and may be preferred over other agents, such as growth factors, peptides and unstable compounds. In the time course study, our ischemia model showed rapid infarct production in first 24 h followed by gradual delayed expansion until 72 h after brain ischemia as reported in usual MCAO (Xu et al., 2006). Although precise mechanism of delayed infarct has remained controversial, previous studies suggested apoptosis plays an important role in the delayed infarct progression, especially in the penumbra (Li et al., 1995). This delayed infarct or apoptotic cell death has been known to diminish the protective effect of the neuroprotective treatment in some case (Dietrich et al., 1993; Kawaguchi et al., 2000). Although the long term outcome of acute COA-Cl treatment was not evaluated in the current study, we demonstrated COA-Cl administration decreased the infarct volume and the number of TUNEL positive cells in ischemic boundary zone at 24 h after brain ischemia.

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Fig. 10 – Linear-regression curve of each histological parameter and the functional outcome after brain ischemia/reperfusion. Linear-regression curve of the retention time in rotarod test on day 13 after brain ischemia/reperfusion and the brain damage (A), the number of BrdUþ/lamininþ cells (B), the number of BrdUþ/DCXþ cells (C), and synaptophysin OD (D) were shown. The result of Rotarod on day 13 after brain ischemia/reperfusion showed relatively strong correlation with the number of BrdUþ/Lamininþ cells and synaptophysin OD compared to the brain damage or the number of BrdUþ/DCXþ cells.

Results from the previous reports and our study suggest that neuroprotective effect of COA-Cl could sustain for a longer period. Furthermore, the peak of apoptosis has been reported to occur 24–48 h after brain ischemia (Xu et al., 2006; Kotani et al., 2008). Thus, delayed COA-Cl treatment might prevent progressive brain damage by inhibiting apoptotic cell death.

3.3. Effects on angiogenesis, neurogenesis, and synaptogenesis Previous studies showed that angiogenesis facilitates functional recovery through an interrelated set of neurorestorative events, including neurogenesis and synaptogenesis (Thored et al., 2007; Beck and Plate, 2009). COA-Cl administration significantly increased the number of BrdUþ/lamininþ cells and increased synaptophysin expression in the penumbral region after brain ischemia. However, the number of BrdUþ/DCXþ cells in the subventricular zone and striatum did not change in brain damage matched subgroup analysis, suggesting COA-Cl stimulates angiogenesis and synaptogenesis but not neurogenesis. In this study, we injected BrdU on days 1–8 after brain ischemia induction to investigate endothelial cell and neural stem cell proliferation during continuous COA-Cl administration. Peak of angiogenesis was reported approximately 7–14 days after brain ischemia (Chu et al., 2012). In contrast, peak of neurogenesis has been reported to occur later than 14 days after brain ischemia (Chu et al., 2012; Kokaia et al., 2006). This different time course

may explain the lack of effect on neurogenesis. Therefore, a longer time course is necessary to conclusively determine the effect of COA-Cl on neurogenesis. Although neurogenesis, angiogenesis and synaptogenesis have been known to interact with each other to promote functional recovery, correlation analysis revealed that angiogenesis and synaptogenesis marker shows more strong correlation with functional outcome than neurogenesis. Few BrdUþ/NeuNþ cells were observed on day 13 after brain ischemia (data not shown), while most newly generated neurons were still immature at this time point indicating that neurogenesis would contribute to later recovery. Synaptic activity couples tightly with local blood flow (Iadecola, 2004), and angiogenesis has pivotal role in neural plasticity and motor learning (Black et al., 1989, 1991; Isaacs et al., 1992), which suggests that COA-Cl-induced angiogenesis may contribute to enhanced synaptogenesis after brain ischemia. In conclusion, our finding demonstrates that (1) COA-Cl is potent neuroprotective agent against both in vitro and in vivo ischemia model, (2) the protective effect of COA-Cl is mediated by ERK1/2 and purinergic receptors, especially P2X receptors at least in vitro, (3) COA-Cl has broad therapeutic time window for transient brain ischemia in vivo and its delayed protection may be mediated by inhibition of the apoptotic cell death, (4) COA-Cl facilitates functional recovery by enhancing angiogenesis and synaptogenesis.

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These findings suggest that COA-Cl could be a novel and potent therapeutic agent for ischemic stroke.

4.

Experimental procedure

4.1.

Chemicals and antibodies

COA-Cl was synthesized as described previously (Bisacchi et al., 1991). Suramin and pyridoxalphosphate-6-azophenyl20 ,40 -disulfonic acid (PPADS) were obtained from Enzo Biochem (New York, NY). PD98059, U73122, wortmannin, and LY294001 were obtained from Cayman Chemical (Ann Arbor, MI). GF109203X was obtained from Wako Pure Chemical Industries (Osaka, Japan). BAPTA-AM was obtained from AAT Bioquest (Sunnyvale, CA). Antibodies for ERK1/2 (mouse) and phospho-ERK1/2 (p-ERK1/2; Thr202/Tyr204; rabbit) were obtained from Cell Signaling Technology (Tokyo, Japan). Doublecortin (DCX, goat) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Bromodeoxyuridine (BrdU, mouse) was purchased from Roche Diagnostics (Tokyo, Japan). Laminin (rabbit) was obtained from Sigma-Aldrich Japan (Tokyo, Japan). Synaptophysin (mouse) was obtained from Merck Millipore (Billerica, MA).

4.2.

In vitro experiments

4.2.1.

Primary cortical neuron cultures

Primary cortical neurons were prepared from rat embryos at 18 days of gestation as described previously (Fujita et al., 2009). Briefly, the meningeal tissue, brain stem, hippocampi, and basal ganglia were removed from the cerebral hemispheres. Collected cortices were incubated for 15 min at 37 1C in Ca2þ and Mg2þ free Hanks’ Balanced Salt Solutions (HBSS; Life Technologies Japan, Tokyo, Japan) containing 0.25% trypsin (Life Technologies Japan) and 0.01% ethylenediaminetetraacetic acid (Sigma-Aldrich Japan). Cells were dissociated by trituration and plated into plastic bottom culture dish (AGC Techno Glass, Funabashi, Japan) coated with poly4 2 D-lysine (10 mg/ml; Sigma-Aldrich Japan) at 5  10 cells/cm in plating medium (Dulbecco’s Modified Eagle Medium Nutrient Mixture F12 [Life Technologies Japan] containing 10% fetal bovine serum [NICHIREI, Tokyo, Japan], and 1% penicillin/ streptomycin [Life Technologies Japan]). Plating medium was replaced after 24 h with neuronal culture medium consisting of Neurobasal medium (Life Technologies Japan) containing 2% B27 (Life Technologies Japan), 0.5% penicillin/streptomycin, and 0.5 mM L-alanyl-L-glutamine (GlutaMAX; Life Technologies Japan). Half of the media was replaced twice weekly, and cells were used in vitro day 12–14.

4.2.2.

Oxygen glucose deprivation

Neuronal cultures were subjected to oxygen-glucose deprivation (OGD) as described previously (Grabb and Choi, 1999), with slight modifications. Cultures were washed twice with balanced salt solution lacking glucose (BSS: 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 1.0 mM NaH2PO4, 26.2 mM NaHCO3, 1.8 mM CaCl2, and 0.01 mM glycine) that was aerated with an anaerobic gas mix (90% N2 and 10% CO2) to

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remove residual oxygen (termed OGD solution). Culture media was replaced with OGD solution, and cultures were transferred to an anaerobic chamber (APM; ASTEC, Kasuya, Japan) containing a gas mixture of 95% N2 and 5% CO2. Cell cultures were incubated at 37 1C for 2 h. Control cell cultures were incubated similarly, but with BSS containing 20 mM glucose in a normal chamber (5% CO2 and atmospheric O2). After incubation for 2 h, the OGD solution was replaced with fresh neural culture media containing vehicle or test drugs and incubated for 24 h until neuronal injury assessment.

4.2.3.

Assessment of neuronal injury

Twenty-four hours after ODG, neuronal injury was evaluated by measuring lactate dehydrogenase (LDH). LDH in the media was examined using the LDH Cytotoxicity Detection Kit (TAKARA, Otsu, Japan). Data were normalized to the amount of LDH released from vehicle-treated cells after OGD (100%) and were corrected for baseline LDH release (0%) measured in control cell cultures for each experiment as described previously (Singer et al., 1999).

4.2.4.

ERK1/2 activation by COA-Cl

ERK activation was evaluated in neuronal cultures. Neurons were subjected to OGD for 2 h as described above. After 2 h, OGD solution was replaced with culture media containing the indicated concentration of COA-Cl for 15 min. ERK inhibition studies were performed with suramin (100 mM), PD98059 (10 mM), PPADS (100 mM), U73122 (5 mM), wortmannin (0.5 mM), LY294002 (20 mM), BAPTA-AM (10 mM) or GF109203X (10 mM). Cells were pretreated with these inhibitors during the last 5 min in OGD solution, and then incubated with COA-Cl for 15 min. After 15 min incubation with COA-Cl, cells were lysed and ERK1/2 activation was detected using western blot analyses.

4.2.5.

Western blot analyses

Cells were rinsed twice with ice-cold phosphate-buffered saline (PBS) and then lysed for 15 min on ice in RIPA buffer (25 mM Tris–Cl pH7.6, 150 mM NaCl, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS]) containing a protease and phosphatase inhibitor cocktail (Roche). Lysates were centrifuged at 15,000g for 15 min at 4 1C to remove insoluble material. Supernatants were mixed with an equal volume of SDS sample buffer (100 mM Tris–HCl pH8.8, 2% SDS, 20% Sucrose, 0.06% bromophenol blue, and 100 mM dithiothreitol) and incubated for 10 min at 90 1C to denature protein. Equal amounts of protein were loaded and separated by 12.5% SDS-polyacrylamide gel electrophoresis and transferred electrophoretically to polyvinylidene difluoride membranes. Membranes were incubated for 1 h in 5% bovine serum albumin (BSA) dissolved in Tris-buffered saline plus 0.01% Tween-20 (TBST) and incubated overnight at 4 1C with the p-ERK1/2 antibody (1:2500 dilution) in blocking buffer with gentle shaking. The membranes were washed 3 times with TBST and incubated for 1 h at room temperature with horseradish peroxidase conjugated secondary antibodies in blocking buffer. Membranes were again washed 3 times with TBST, followed by incubation with enhanced chemiluminescence substrate (Western Lightning ECL Pro, PerkinElmer Japan, Yokohama, Japan). Chemiluminescence was detected

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using a charge-coupled device camera image system (LAS4000 mini; GE Healthcare Japan, Tokyo, Japan). Membranes were stripped using a commercially available stripping buffer (Thermo Fisher Scientific, Yokohama, Japan) and reprobed with ERK1/2 antibodies as loading controls. Quantitative data show the mean7SD from four independent experiments.

4.3.

In vivo experiments

4.3.1.

Animals

All animal experiments were approved by the Animal Research Committee of Kawasaki Medical School and conducted according to the ‘‘Guide for Care and Use of Laboratory Animals’’ of Kawasaki Medical School. A total of 88 male Sprague-Dawley rats (weighing 250–320 g; CREA, Tokyo, Japan) were used in the in vivo experiments. The rats were housed under a 12-h light/dark cycle and had access to food and water ad libitum. All animals were randomized in each experiment. The behavior tests and infarct volume measurements were performed by an individual that was blinded to treatment with either saline or COA-Cl.

4.3.2.

Rat model of transient focal cerebral ischemia

Transient focal cerebral ischemia was induced using the suture method for middle cerebral artery occlusion (MCAO) (Miyamoto and Auer, 2000), combined with bilateral common carotid artery occlusion to reproduce stable deficits in behavior tests (Petullo et al., 1999). Rats were anesthetized with 2% isoflurane in 70% N2O and 30% O2 under spontaneous respiration. Rectal temperature was maintained at 37.570.5 1C using a heating pad and heating lamp (ATB1100, NIHON KOHDEN Corporation, Tokyo). The left femoral artery was cannulated (polyethylene tube SP31, NATSUME SEISAKUSYO Co Ltd, Tokyo) and connected to a pressure transducer (RM600, NIHON KOHDEN Corporation, Tokyo, Japan). The rats were not cannulated in the continuous administration study to avoid adverse effects in the rotarod tests. Body temperature, heart rate and mean arterial blood pressure were monitored continuously. Arterial pH, PaCO2, and PaO2 were measured (850ASH, Bayer medical Ltd, Tokyo, Japan) pre- and post-ischemia and 30 min after the onset of ischemia. After a neck incision, the common carotid arteries (CCAs) were isolated, and the left CCA was occluded with a microaneurysm clip. A blunt tip 3-0 nylon suture was inserted into the left external carotid artery and advanced into the internal carotid artery until faint resistance was encountered. The right CCA was occluded with a microaneurysm clip. After occlusion for 60 min, the microaneurysm clips and suture were removed to restore blood flow.

4.3.3.

Acute administration of COA-Cl after brain ischemia

Rats were intracerebroventricularly administered COA-Cl (6, 15, or 30 mg/kg, n¼ 6), or the same volume of saline (n¼ 6) 5 min after the onset of reperfusion. A cranial burr hole (0.8 mm) was drilled (0.8 mm posterior, 1.8 mm lateral to the bregma) before induction of ischemia, and a 24-gauge needle was inserted to 4.5 mm deep from the skull surface after ischemia. COA-Cl or saline was injected at 1 ml/min for 5 min. Needle was stayed for further 5 min after the end of

injection to prevent the leakage of drug. Twenty-four hours after brain ischemia, the rats were deeply anesthetized with pentobarbital (IP, 80 mg/kg) and decapitated. The brains were coronally sectioned into seven 2-mm-thick slices and stained with a 1% solution of triphenyltetrazolium chloride (TTC). The images were analyzed using the NIH image program, and areas were multiplied by the distance between sections to obtain infarct volume, and edema (left hemisphereright hemisphere) and corrected infarct volumes (infarct volumeedema) were calculated. For the TUNEL staining, rats were treated with saline or 15 mg/kg (n¼ 7) of COA-Cl (n¼ 7) as describe above. Following 24 h reperfusion, rats were perfused with 4% paraformaldehyde and placed in the same fixative overnight. The brains were immersed in 10% sucrose for 1 day, 20% sucrose for 1 day and 30% sucrose for 3 days. After cryoprotection with 30% sucrose, the brains were sectioned using a cryostat into 10mm-thick sections at 0.92, 0.56 and 0.20 mm anterior to the bregma.

4.3.4. Continuous COA-Cl administration and BrdU labeling after brain ischemia One day after induction of brain ischemia/reperfusion, rats were anesthetized and implanted with osmotic mini-pumps (Model 2001, Alzet, Cupertino, CA). COA-Cl (0.5 mg/ml) was administered at 1.5 mg/kg/h for 7 days (n¼ 7). Control rats received saline at the same rate for the same period (n¼7). BrdU (50 mg/kg) was given once daily by IP injection to both the groups for the time period. On day 13 after brain ischemia/reperfusion, the rats were deeply anesthetized with pentobarbital (IP, 80 mg/kg) and perfused with 4% paraformaldehyde. After cryoprotection with 30% sucrose, the brains were sectioned using a cryostat into 16-mmthick sections. For infarct volume measurements, sections were prepared from the same locations as in the acute administration study. Sections were stained with hematoxylin and eosin (HE), and then NIH image program was used to obtain infarct volume, atrophy (right hemisphereleft hemisphere), healthy tissue (left hemisphereinfarct volume), and total brain damage (infarct volumeþatrophy).

4.3.5.

Time course study of infarct volume

Progress of pathological changes in our brain ischemia model was examined by TTC staining method. Rats were subjected to brain ischemia as describe above and sacrificed at 24 h (n ¼7), 48 h (n ¼6), or 72 h (n ¼7) after brain ischemia. The brains were sectioned and stained with TTC, and the volume of edema, corrected infarct and the healthy tissue were measured.

4.3.6.

Behavior tests

For the acute administration study, clinical scoring was performed at 24 h after brain ischemia. For the continuous administration study, rats were subjected to clinical scoring and rotarod tests before brain ischemia and 1, 3, 6, and 13 days after ischemia. Neurological deficits were graded according to the clinical scoring criteria described previously (Petullo et al., 1999), with slight modifications. Clinical scoring criteria consisted of 6 subtests; these included tests for forelimb flexion, twisting, resistance to lateral push, circling, forelimb placement, and

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hindlimb placement. In each test, the degree of neurological deficits was graded from 0 to 1 (0¼ no deficit, 0.5¼ mild, 1¼ moderate to severe), and total clinical score ranged from 0 to 6. Rats were excluded if total clinical score was o2. Coordinated movements were further evaluated using the rotarod (Muromachi Kikai, Tokyo, Japan) as described previously (Lin et al., 2011). The animals were placed on a rotating wheel accelerating from 4 rounds per min (rpm) to 40 rpm within 5 min. The time the rats remained on the rotating wheel was measured and averaged from 3 repeated trials. The rats were habituated for 3 days before surgery and the base lines were determined from the measurement the day before brain ischemia. The data are shown as the percentage of retention time that recorded before brain ischemia.

4.3.7.

Regional cerebral blood flow (rCBF) measurement

Cortical rCBF was continuously monitored using a laser Doppler probe (FLO C1, OMEGAWAVE, Tokyo, Japan ) with a 0.5-mm diameter to evaluate the rCBF changes after COA-Cl treatment. rCBF monitoring was performed in a total of 16 animals: 8 saline-treated rats and 8 COA-Cl-treated rats (15 mg/kg). After two cranial burr hole (0.6 mm) were drilled (0.8 mm posterior, 1.8 mm lateral to the bregma for drug administration, 1.0 mm posterior, 5.0 mm lateral to the bregma for rCBF measurement), Bilateral CCAs and left external carotid artery were isolated and 5 cm length blunt tip 3-0 nylon suture was inserted into the left external carotid artery and advanced only 1 cm. Nylon suture was loosely fixed by ligature, and then the rat was placed in a stereotaxic frame in prone position. Cortical rCBF was monitored 10 min before brain ischemia to establish the baseline values. Following the left CCA occlusion with a microaneurysm clip, MCA were occluded by advancing the nylon suture until steep decline of rCBF was observed. Right CCA was occluded and cortical rCBF was continuously monitored through the whole period of ischemia. After 1 h of ischemia, the filament was withdrawn, and recovery of cortical blood flow was observed for 60 min. The changes in cortical CBF were expressed as percentage of the baseline level.

4.3.8.

TUNEL staining

To detect DNA fragmentation, TUNEL assay was performed with ApopTag Plus Peroxidase In situ Apoptosis Detection Kit (Merck Millipore) according to the manufacturer’s instruction as described previously (Miyamoto et al., 2003). Briefly, sections were permeabilized with 0.1% Triton X-100. For exposing the DNA, the sections were boiled for 2 cycles of 5 min each in microwave oven and then treated with proteinase K (20 mg/ml, R&D system, Minneapolis) for 15 min. After quenching by 3.0% hydrogen peroxide, sections were labeled with TdT reaction solution. They were labeled with AntiDigoxignenin-Peroxidase, and peroxidase was detected with diaminobenzidine. Four pictures were taken from ischemic border of each section using  40 objectives and the number of TUNEL positive cells was counted (3 sections/rat, n ¼7 for each group).

4.3.9.

Immunohistochemistry

Rat brain sections were washed twice with PBS. For BrdU straining, sections were boiled for 10 min in 0.01 M citrate buffer

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ion a microwave (200 W) to denature DNA. The sections were permeabilized for 15 min with 0.1% Triton X-100 and then incubated for 1 h with blocking buffer containing 2% BSA, 0.1% Triton X-100 in 0.01 M PBS. Sections were incubated overnight at 4 1C with the laminin (1:1000), DCX (1:400), BrdU (1:400), or synaptophysin (1:1000) antibodies and dissolved in 0.01 M PBS containing 1% BSA. The sections were washed 3 times with PBS and incubated with Alexa Fluor-conjugated secondary antibodies (1:1000, Molecular Probes) at room temperature for 1 h.

4.3.10.

Image analyses

Angiogenesis, synaptogenesis, and neurogenesis were screened in 3 brain sections at 0.92, 0.56 and 0.20 mm anterior to the bregma (Arvidsson et al., 2002). To quantify angiogenesis after brain ischemia, 2 images of the cortical penumbra region were acquired from each section using epifluorescence microscopy (  20 objective; BX61, OLYMPUS Corporation, Tokyo, Japan) and the number of BrdU and laminin double-labeled cells were counted. The ischemic border zone is defined as the area surrounding the lesion, which morphologically differs from the surrounding normal tissue (Nedergaard et al., 1987). Cells were considered double-labeled if a cytoplasmic marker surrounded a nuclear marker. For neurogenesis quantification, images of the subventricular zone and striatum were acquired using epifluorescence microscopy (  20 objective) and the number of all BrdU and DCX double-labeled cells in the subventricular zone and striatum were counted. The accuracy of double-labeled cell counting in angiogenesis and neurogenesis studies was confirmed using confocal laser scanning microscopy (FV1000-D, OLYMPUS) in some sections. To quantify synaptogenesis after brain ischemia, 4 images of the cortical ischemic penumbra region were acquired from each section using epifluorescence microscopy (  100 objective), and the optical density of the synaptophysin immunoreactive area in each image was determined using NIH image analysis program.

4.4.

Statistical analyses

All data except for clinical data are reported as mean7SD. Comparisons were made using ANOVA, and multiple comparisons were performed with post hoc Tukey’s test or Student’s ttest. Clinical score was analyzed with Kruskal–Wallis or Mann–Whitney test and data are reported as medians and IQR. For rotarod test, data were subjected to two-way repeated measures ANOVA, and multiple comparisons were performed post hoc using Bonferroni’s method. The correlation analyses were performed with Pearson’s linear-regression method. A statistically significant difference was defined as a P value less than 0.05. All statistical analyses were performed using GraphPad Prism (GraphPad, La Jolla, CA).

Acknowledgment This work was partially funded by the KAWASAKI foundation for Medical Science and Medical Welfare, as well as the Ryobi Teien Memorial Foundation.

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Appendix A.

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Supporting information

Supplementary data associated with this article can be found in the online version at: http://dx.doi.org/10.1016/j.brainres. 2013.02.009.

references

Allan, L.A., Morrice, N., Brady, S., Magee, G., Pathak, S., Clarke, P.R., 2003. Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK. Nat. Cell Biol. 5, 647–654. Arvidsson, A., Collin, T., Kirik, D., Kokaia, Z., Lindvall, O., 2002. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat. Med. 8, 963–970. Baron, J.C., von Kummer, R., del Zoppo, G.J., 1995. Treatment of acute ischemic stroke. Challenging the concept of a rigid and universal time window. Stroke 26, 2219–2221. Beck, H., Plate, K.H., 2009. Angiogenesis after cerebral ischemia. Acta Neuropathol. 117, 481–496. Bisacchi, G.S., Braitman, A., Cianci, C.W., Clark, J.M., Field, A.K., Hagen, M.E., Deborah, R., Hockstein, Malley, M.F., Toomas, M., 1991. Synthesis and antiviral activity of enantiomeric forms of cyclobutyl nucleoside analogues. J. Med. Chem. 34, 1415–1421. Black, J.E., Zelazny, A.M., Greenough, W.T., 1991. Capillary and mitochondrial support of neural plasticity in adult rat visual cortex. Exp. Neurol. 111, 204–209. Black, J.E., Polinsky, M., Greenough, W.T., 1989. Progressive failure of cerebral angiogenesis supporting neural plasticity in aging rats. Neurobiol. Aging 10, 353–358. Chu, M., Hu, X., Lu, S., Gan, Y., Li, P., Guo, Y., Zhang, J., Chen, J., Gao, Y., 2012. Focal cerebral ischemia activates neurovascular restorative dynamics in mouse brain. Front. Biosci. 4, 1926–1936. Dietrich, W.D., Busto, R., Alonso, O., Globus, M.Y., Ginsberg., M.,.D., 1993. Intraischemic but not postischemic brain hypothermia protects chronically following global forebrain ischemia in rats. J. Cereb. Blood Flow Metab. 13, 541–549. Fujita, T., Tozaki-Saitoh, H., Inoue, K., 2009. P2Y1 receptor signaling enhances neuroprotection by astrocytes against oxidative stress via IL-6 release in hippocampal cultures. Glia 57, 244–257. Grabb, M.C., Choi, D.W., 1999. Ischemic tolerance in murine cortical cell culture: critical role for NMDA receptors. J. Neurosci. 19, 1657–1662. Gu, W., Brannstrom, T., Jiang, W., Bergh, A., Wester, P., 2001. Vascular endothelial growth factor-A and -C protein upregulation and early angiogenesis in a rat photothrombic ring stroke model with spontaneous reperfusion. Acta Neuropathol. 102, 216–226. Harada, T., Morooka, T., Ogawa, S., Nishida, E., 2001. RK induces p35, a neuron-specific activator of Cdk5, through induction of Egr1. Nat. Cell Biol. 3, 453–459. Hetman, M., Kanning, K., Cavanaugh, J.E., Xia, Z., 1999. Neuroprotection by brain-derived neurotrophic factor is mediated by extracellular signal-regulated kinase and phosphatidylinositol 3-kinase. J. Biol. Chem. 274, 22569–22580. Iadecola, C., 2004. Neurovascular regulation in the normal brain and in Alzheimer’s disease. Nat. Rev. Neurosci. 5, 347–360. Isaacs, K.R., Anderson, B.J., Alcantara, A.A., Black, J.E., Greenough, W.T., 1992. Exercise and the brain: angiogenesis in the adult rat cerebellum after vigorous physical activity and motor skill learning. J. Cereb. Blood Flow Metab. 12, 110–119. Kawaguchi, M., Kimbro, J.R., Drummond, J.C., Cole, D.J., Kelly, P.J., Patel, P.M., 2000. Isoflurane delays but does not prevent

cerebral infarction in rats subjected to focal ischemia. Anesthesiology 92, 1335–1342. Kilic, E., Kilic, U., Soliz, J., Bassetti, C.L., Gassmann, M., Hermann, D.M., 2005. Brain-derived erythropoietin protects from focal cerebral ischemia by dual activation of ERK-1/-2 and Akt pathways. FASEB J. 19, 2026–2028. Kokaia, Z., Thored, P., Arvidsson, A., Lindvall, O., 2006. Regulation of stroke-induced neurogenesis in adult brain—recent scientific progress. Cereb. Cortex 16, 162–167. Kong, Q., Wang, M., Liao, Z., Camden, J.M., Yu, S., Simonyi, A., Sun, G.Y., Gonzalez, F.A., Erb, L., Seye, C.I., Weisman, G.A., 2005. P2X(7) nucleotide receptors mediate caspase-8/9/3dependent apoptosis in rat primary cortical neurons. Purinergic Signal 1, 337–347. Kotani, Y., Nakajima, Y., Hasegawa, T., Satoh, M., Nagase, H., Shimazawa, M., Yoshimura, S., Iwama, T., Hara, H., 2008. Propofol exerts greater neuroprotection with disodium edetate than without it. J. Cereb. Blood Flow Metab. 28, 354–366. Krupinski, J., Kaluza, J., Kumar, P., Kumar, S., Wang, J.M., 1994. Role of angiogenesis in patients with cerebral ischemic stroke. Stroke 25, 1794–1798. Li, Y., Chopp, M., Jiang, N., Yao, F., Zaloga, C., 1995. Temporal profile of in situ DNA fragmentation after transient middle cerebral artery occlusion in the rat. J. Cereb. Blood Flow Metab. 15, 389–397. Lin, Y.C., Ko, T.L., Shih, Y.H., Lin, M.Y., Fu, T.W., Hsiao, H.S., Hsu, J.Y., Fu, Y.S., 2011. Human umbilical mesenchymal stem cells promote recovery after ischemic stroke. Stroke 42, 2045–2053. Luciano, F., Jacquel, A., Colosetti, P., Herrant, M., Cagnol, S., Pages, G., Auberger, P., 2003. Phosphorylation of Bim-EL by Erk1/2 on serine 69 promotes its degradation via the proteasome pathway and regulates its proapoptotic function. Oncogene 22, 6785–6793. Miyamoto, O., Auer, R.N., 2000. Hypoxia, hyperoxia, ischemia, and brain necrosis. Neurology 54, 362–371. Miyamoto, O., Tamae, K., Kasai, H., Hirakawa, H., Hayashida, Y., Konishi, R., Itano, T., 2003. Suppression of hyperemia and DNA oxidation by indomethacin in cerebral ischemia. Eur. J. Pharmacol. 459, 179–186. Nedergaard, M., Gjedde, A., Diemer, N.H., 1987. Hyperglycaemia protects against neuronal injury around experimental brain infarcts. Neurol. Res. 9, 241–244. Ortega, F., Pe´rez-Sen, R., Delicado, E.G., Miras-Portugal, M.T., 2009. P2  7 nucleotide receptor is coupled to GSK-3 inhibition and neuroprotection in cerebellar granule neurons. Neurotox. Res. 15, 193–204. Ortega, F., Pe´rez-Sen, R., Delicado, E.G., Teresa Miras-Portugal, M., 2011. ERK1/2 activation is involved in the neuroprotective action of P2Y13 and P2  7 receptors against glutamate excitotoxicity in cerebellar granule neurons. Neuropharmacology 61, 1210–1221. Petullo, D., Masonic, K., Lincoln, C., Wibberley, L., Teliska, M., Yao, D.L., 1999. Model development and behavioral assessment of focal cerebral ischemia in rats. Life Sci. 64, 1099–1108. Singer, C.A., Figueroa-Masot, X.A., Batchelor, R.H., Dorsa, D.M., 1999. The mitogen-activated protein kinase pathway mediates estrogen neuroprotection after glutamate toxicity in primary cortical neurons. J. Neurosci. 19, 2455–2463. Sugiyama, T., Oku, H., Shibata, M., Fukuhara, M., Yoshida, H., Ikeda, T., 2010. Involvement of P2  7 receptors in the hypoxiainduced death of rat retinal neurons. Invest. Ophthalmol. Vis. Sci. 51, 3236–3243. Sun, Y., Jin, K., Xie, L., Childs, J., Mao, X.O., Logvinova, A., Greenberg, D.A., 2003. VEGF-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia. J. Clin. Invest. 111, 1843–1851. Thored, P., Wood, J., Arvidsson, A., Cammenga, J., Kokaia, Z., Lindvall, O., 2007. Long-term neuroblast migration along blood

brain research 1506 (2013) 115–131

vessels in an area with transient angiogenesis and increased vascularization after stroke. Stroke 38, 3032–3039. Tsukamoto, I., Sakakibara, N., Maruyama, T., Igarashi, J., Kosaka, H., Kubota, Y., Tokuda, M., Ashino, H., Hattori, K., Tanaka, S., Kawata, M., Konishi, R., 2010. A novel nucleic acid analogue shows strong angiogenic activity. Biochem. Biophys. Res. Commun. 399, 699–704. Wang, Y., Zhang, Z.G., Rhodes, K., Renzi, M., Zhang, R.L., Kapke, A., Lu, M., Pool, C., Heavner, G., Chopp, M., 2007. Post-ischemic treatment with erythropoietin or carbamylated erythropoietin reduces infarction and improves neurological outcome in a rat model of focal cerebral ischemia. Br. J. Pharmacol. 151, 1377–1384. Wei, L., Erinjeri, J.P., Rovainen, C.M., Woolsey, T.A., 2001. Collateral growth and angiogenesis around cortical growth. Stroke 32, 2179–2184.

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Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J., Greenberg, M.E., 1995. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270, 1326–1331. Xiao, Z., Kong, Y., Yang, S., Li, M., Wen, J., Li, L., 2007. Upregulation of Flk-1 by bFGF via the ERK pathway is essential for VEGFmediated promotion of neural stem cell proliferation. Cell Res. 17, 73–79. Xu, X.H., Zhang, S.M., Yan, W.M., Li, X.R., Zhang, H.Y., Zheng, X.X., 2006. Development of cerebral infarction, apoptotic cell death and expression of X-chromosome-linked inhibitor of apoptosis protein following focal cerebral ischemia in rats, Life Sci. 78, 704–712. Zhang, F., Wang, S., Signore, A.P., Chen, J., 2007. Neuroprotective effects of leptin against ischemic injury induced by oxygenglucose deprivation and transient cerebral ischemia. Stroke 38, 2329–2336.