Treatment with edaravone attenuates ischemic brain injury and inhibits neurogenesis in the subventricular zone of adult rats after focal cerebral ischemia and reperfusion injury

Neuroscience 201 (2012) 297–306

TREATMENT WITH EDARAVONE ATTENUATES ISCHEMIC BRAIN INJURY AND INHIBITS NEUROGENESIS IN THE SUBVENTRICULAR ZONE OF ADULT RATS AFTER FOCAL CEREBRAL ISCHEMIA AND REPERFUSION INJURY P. ZHANG,a* W. LI,a,b L. LI,a,b N. WANG,a,b X. LI,a,b M. GAO,a,b J. ZHENG,a,b S. LEI,a,b X. CHEN,b H. LUb AND Y. LIUb

Stroke is the second leading cause of death in China, with ischemic stroke being the most common type (Shi et al., 2009). Minimization of the infarct area and generation of new neuronal cells in the injured brain are considered important strategic approaches for stroke treatment. The subventricular zone (SVZ), which is located in the lateral wall lining the lateral ventricle, harbors the largest population of neural stem cells (NSCs) that are capable of generating new neurons, astrocytes, and oligodendrocytes in rodents (Emsley et al., 2005), monkeys (Kornack and Rakic, 2001), and humans (Bernier et al., 2000). Proliferating SVZ cells normally migrate through the SVZ and along the rostral migratory stream (RMS) into the olfactory bulb and are finally incorporated as olfactory interneurons (Emsley et al., 2005). Increasing experimental evidence indicates that several pathological conditions such as cerebral ischemia/hypoxia, traumatic and primary degenerative diseases are known to increase neurogenesis in the SVZ of adult brains (Schmidt and Reymann, 2002; Im et al., 2010; Ohira, 2011). Hypoxia-inducible factor 1␣ (HIF-1␣) is an important transcriptional factor implicated in many cerebrovascular pathological disorders. Several studies indicate that the hypoxia-inducible factor 1 (HIF-1) signaling pathway appears important for stroke-induced SVZ neurogenesis (Androutsellis-Theotokis et al., 2006; Zhang et al., 2006 –2007; Panchision, 2009). HIF-1␣ regulates neurogenesis through increase of vascular endothelial growth factor (VEGF) and erythropoietin (EPO) (Schölzke and Schwaninger, 2007; Tang et al., 2010). VEGF stimulates neural stem cell proliferation and is an attractive guidance cue for the migration of SVZ neural progenitors in vitro (Schänzer et al., 2004; Zhang et al., 2003). Moreover, evidence has been accumulating for the involvement of reactive oxygen species (ROS) in the activation of signaling components upstream of HIF-1␣ such as hydroxylases and kinases (Hwang et al., 2008; Koshikawa et al., 2009). Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a novel free radical scavenger. It attenuates ischemic brain injury in patients and animal models (Watanabe et al., 2008; Yamamoto et al., 2009). Edaravone scavenges ROS, suppresses the accumulation of HIF-1␣ in the nuclei, and reduces HIF-1␣ binding to VEGF promoter in human astrocytes exposed to hypoxia (Ishikawa et al., 2007; Watanabe et al., 2008). However, it is still unclear whether edaravone plays a role in the neurogenesis following cerebral ischemia. In this study, we used a focal cerebral

a Department of Anesthesiology, Second Affiliated Hospital of Xi’an Jiaotong University School of Medicine, #157 West 5 road, Xi’an, Shaanxi 710004, China b Institute of Neurobiology, National Key Academic Subject of Physiology of Xi’an Jiaotong University School of Medicine, #76 Yanta West Road, Xi’an, Shaanxi 710061, China

Abstract—Edaravone is a novel free radical scavenger that is clinically employed in patients with acute cerebral infarction. However, its effect on stroke-induced subventricular zone (SVZ) neurogenesis is largely unknown. In this study, we investigated the effect and underlying mechanism of edaravone administration on SVZ neurogenesis using a rat model of cerebral ischemia-reperfusion injury. Male Sprague–Dawley rats (200 –250 g) were divided into sham operated (nⴝ15), control (nⴝ50), and edaravone-treated (nⴝ50) groups. Rats in the control and edaravone-treated groups underwent 90 min of middle cerebral artery occlusion (MCAO) following reperfusion. Immediately and 12 h after MCAO, the rats received either normal saline (control group) or edaravone (edaravone-treated group) intraperitoneally. 5-bromo-2-deoxyuridine (BrdU) was used to label proliferating cells. Six, 12, and 24 hours after ischemia, reactive oxygen species (ROS) generation, hypoxia-inducible factor 1␣ (HIF-1␣), and vascular endothelial growth factor (VEGF) protein levels in ischemic ipsilateral SVZ were determined. Immunohistochemistry staining for BrdU and doublecortin (DCX) was performed at 1, 4, and 7 days after ischemia. Treatment with edaravone not only mitigated cerebral infarct size (P<0.05) and neurological defects (P<0.05), but also decreased cell proliferation and neural progenitor cells in the ischemic ipsilateral SVZ (P<0.05). Additionally, edaravone reduced effectively ROS generation and HIF-1␣ as well as VEGF protein levels in the ischemic ipsilateral SVZ (P<0.05). These findings indicate that administration with edaravone, via repressing HIF-1␣ signaling pathway, inhibits SVZ neurogenesis in rats after cerebral ischemia-reperfusion injury. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: neurogenesis, reactive oxygen species, hypoxiainducible factor 1␣, cerebral ischemia, reperfusion injury. *Corresponding author. Tel: ⫹86-29-87679635; fax: ⫹86-29-87678223. E-mail address: [email protected] (P. Zhang). Abbreviations: ANOVA, analysis of variance; BrdU, 5-bromo-2-deoxyuridine; DCX, doublecortin; EPO, erythropoietin; HIF-1␣, hypoxiainducible factor 1␣; MCAO, middle cerebral artery occlusion; mNSS, modified neurological severity score; NSC, neural stem cell; NSCs, neural stem cells; NSPCs, neural stem/progenitor cells; RMS, rostral migratory stream; ROS, reactive oxygen species; SVZ, subventricular zone; TRITC, tetramethyl rhodamine isothiocyanate; VEGF, vascular endothelial growth factor. 0306-4522/12 $36.00 © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2011.11.005

297

298

P. Zhang et al. / Neuroscience 201 (2012) 297–306

ischemia-reperfusion model to explore the effects of edaravone administration on neurogenesis in the ipsilateral SVZ.

EXPERIMENTAL PROCEDURES Animal modeling All animals were provided by Experimental Animal Center of Xi’an Jiaotong University School of Medicine (Certificate No. 229601018). All studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23). Experimental protocols were approved by the Animal Care and Use Regulation of Xi’an Jiaotong University School of Medicine. Male adult Sprague–Dawley rats, weighing 200 –250 g, were maintained on a 12-h light/dark cycle with free access to food and water. Rats in the edaravone group (n⫽50) were treated with intraperitoneal injections (IP) of edaravone (3 mg/kg, immediately and 12 h after middle cerebral artery occlusion (MCAO), Boda, Jilin, China). Rats in the control group (n⫽50) received an intraperitoneal injection of normal saline at a volume similar to that used in the edaravone group. Ischemia was induced by the intraluminal vascular occlusion method as described previously. Briefly, rats were anesthetized with pentobarbital sodium (40 mg/kg) intraperitoneally and a 3-0 surgical monofilament nylon suture with rounded tip was introduced into the left internal carotid through the arteriotomy and advanced 16.5–17.5 mm past the carotid bifurcation. Ninety minutes later, the monofilament nylon suture was withdrawn for reperfusion. Rats in the sham-operated group (n⫽15) received the same surgical procedures except that the artery was not incised and occluded. To label the population of proliferating cells during 7 days of stroke, cumulative BrdU labeling was employed; briefly, BrdU (50 mg/kg, Sigma-Aldrich, USA) was injected intraperitoneally at the onset of ischemia and then daily for 1, 4 or 7 consecutive days. The rats were killed 2 h after the final injection (Zhang et al., 2001; Lee et al., 2007). Neurological function was assessed by modified neurological severity score (mNSS) (Zhang et al., 2009). At 24 h (n⫽5 each) after ischemia, 2,3,5-triphenyl tetrazolium chloride (TTC) staining was performed on 2-mm-thick coronal brain sections throughout the brain to evaluate the infarct size as described previously (Hsieh et al., 2006).

Immunohistochemistry One, 4 and 7 days after ischemia, rats (n⫽5 per time point) were anesthetized with pentobarbital and perfused transcardially with normal saline followed by 4 % paraformaldehyde in PBS. The brains were removed, and tissue from bregma ⫹0.2 mm to bregma ⫺4.0 mm was taken and fixed in 4% paraformaldehyde overnight, then cut into 20-␮m-thick coronal sections on a cryostat microtome (HM505 E, Microm, Walldorf, Germany). BrdU staining was performed according to a previous report (Zhang et al., 2011). The sections were incubated in 1% H2O2 in PBS for 20 min and in blocking solution (2% goat serum, 0.3% Triton X-100, and 0.1% bovine serum albumin in PBS) for 30 min at room temperature before being treated overnight at 4 °C with the primary antibodies: rabbit-anti-BrdU (1:1,000, Abcam, Cambridge, UK), rat-anti-BrdU (1:1,000, Abcam, UK), and rabbit polyclonal anti-rat doublecortin (DCX) (1:300, Sigma-Aldrich, USA), then washed with PBS/0.3% Triton X-100. For enzyme immunohistochemistry, the sections were incubated with biotinylated goat anti-rabbit immunoglobulin IgG secondary antibody for 2 h at room temperature, rinsed, and placed in avidin-peroxidase conjugate solution for 1 h. The horseradish peroxidase was detected with 0.05% DAB (Sigma) and 0.03% H2O2. For double immunofluorescence, the sections were incubated with fluorescein isothiocyanate

(FITC)- and tetramethyl rhodamine isothiocyanate (TRITC)conjugated IgG for 2 h at room temperature. Sections were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA), and fluorescence signals were detected with a laser confocal microscope (TSC SP2, Leica) at excitation/emission wavelengths of 535 nm/565 nm (TRITC, red) and at 470 nm/ 505 nm (FITC, green). The sections incubated with PBS instead of the primary antibodies were taken as negative controls.

Western blotting To detect the protein expressions of HIF-1␣ and VEGF in ischemic ipsilateral SVZ, five rats per time point after 6, 12, and 24 h of ischemia were decapitated, and the brains were removed quickly. The SVZ (bregma ⫹0.2 mm to bregma ⫺4.0 mm) of ischemic hemispheres was cut into small pieces and homogenized in cold protein extraction buffer (150 mM NaCl, 50 mM Tris–HCl, pH 7.6, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 20 mM Na4P2O7, 10% glycerol, Pierce, Rockford, IL, USA), centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was collected and measured by BCA assay. Equal amounts of protein (50 ␮g) were electrophoresed on a 12% SDS-PAGE and transferred to nitrocellulose membrane (0.45 ␮m, Millipore, USA) for 2 h at 1 mA/cm2. After incubation with 10% non-fat milk for 2 h at room temperature to mask the non-specific binding site of IgG, the membranes were incubated overnight at 4 °C with rabbit polyclonal anti-HIF-1␣ antibody (reacts with human, mouse, rat, chicken, monkey; 1:1,000, Abcam, Cambridge, UK), rabbit polyclonal anti-VEGF antibody (reacts with human, mouse, rat; 1:1,000, Abcam, Cambridge, UK), and mouse monoclonal anti␤-actin antibody (1:10,000, Abcam, UK), followed by incubation with anti-rabbit horseradish peroxidase-conjugated IgG (1: 5,000, Santa Cruz, CA, USA) and anti-mouse horseradish peroxidase-conjugated IgG (1:1,000, Pierce Biotechnology, USA) for 2 h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescent substrate (Thermo Scientific Pierce, USA) using horseradish peroxidase-labeled secondary antibodies (1:5,000, Santa Cruz, CA, USA). The housekeeping protein ␤-actin was used as a control and tested simultaneously by mouse monoclonal anti-␤-actin antibody (1: 10,000, Sigma-Aldrich, USA). The luminescent signal is detected by the CCD camera and transmitted to the controller unit, and the data are sent to the computer for analysis and documentation.

In situ superoxide detection Rats were anesthetized by pentobarbital (40 mg/kg, IP) and sacrificed 6, 12, and 24 h after the onset of ischemia (n⫽5 per time point). The brains were removed and frozen immediately by liquid nitrogen. Twenty-five-␮m-thick coronal sections, spaced 200 ␮m apart, from bregma ⫹0.2 mm to bregma ⫺4.0 mm were prepared for analysis in situ. The sections were incubated with dihydroethidium (DHE; 5 ␮mol/L, Sigma-Aldrich, USA) in PBS for 30 min at 37 °C in a humidified chamber protected from light. DHE is oxidized on reaction with SO2⫺ to ethidium bromide which, in turn, binds to DNA in the nucleus and emits red fluorescence (Shichinohe et al., 2004; Yamamoto et al., 2009). The red fluorescence was detected through a 580-nm long-pass filter using fluorescence microscopy (BX51, Olympus, Japan) and was digitally photographed using a cooled CCD (DP71) camera mounted to the microscope (BX51, Olympus, Japan). On each coronal section (n⫽4 in each rat), the intensity of red fluorescence in ischemic ipsilateral SVZ was semi-quantitatively analyzed using Image-Pro Plus 5.0 for Windows (Media Cybernetics, MD, USA). The fluorescence signal was also measured in the same regions of the

P. Zhang et al. / Neuroscience 201 (2012) 297–306

299

Fig. 1. Representative pictures of ischemic injury and sham-operated brains. (A, B) TTC staining of the brain from rats treated with either control (A) or edaravone (B). Bar⫽1 cm. (C, D) Immunofluorescence staining of BrdU (C) or DCX (D) in the SVZ of sham-operated rats. Bar⫽50 ␮m. (E) DHE staining in the SVZ of sham-operated rats. Bar⫽80 ␮m.

contralateral SVZ. The ratio of the ipsilateral to the contralateral intensity was calculated to assess the production of superoxide in studied regions.

Cell counts The sections were examined under a light microscope (BX51, Olympus, Japan). Defined regions of interest within the SVZ were counted blindly in four 20-␮m coronal sections per animal, spaced 200 ␮m apart. Cells were counted under high-power (⫻20 objective) using Image-Pro Plus 5.0 for Windows (Media Cybernetics, MD, USA). Cell number in the regions of interest was averaged to obtain a mean value for each animal (Lee et al., 2007). BrdU/ DCX-stained sections were analyzed under laser confocal microscopy (TSC SP2, Leica).

Statistical analysis Statistical analysis was conducted using the SPSS for Windows 10.0 software (version 10.0; SPSS Inc., Chicago, IL, USA). To determine the difference in the number of BrdU-positive cells or DCX-positive cells, a one-way analysis of variance (ANOVA) was performed; subsequently, a Bonferroni-Tamhane post hoc test was used to assess the differences between different time points. An independent-sam-

ple t-test was used to assess the differences between the groups. All the data in this study are presented as mean⫾SD. A two-tailed probability value of P⬍0.05 was considered significant.

RESULTS Edaravone decreased infarct size and neurological deficits After ischemia-reperfusion, a white-stained infarct area was observed in both the edaravone group (Fig. 1A) and the control group (Fig. 1B). Percentage of the cerebral infarction area was 35.6⫾4.1% in the control group. When treated with 3.0 mg/kg of edaravone immediately and 12 h after the onset of ischemia, the percentage of cerebral infarction area was 21.8⫾3.2% (P⬍0.05 compared with control). Furthermore, the mNSS was significantly lower in the edaravone-treated rats at days 0.5 to14 after ischemia, compared with the control group (P⬍0.05; Fig. 3A). However, the mNSS was not significantly different at days 21–28 after ischemia between the two groups (P⬎0.05; Fig. 3A).

300

P. Zhang et al. / Neuroscience 201 (2012) 297–306

Fig. 2. Changes in BrdU-labeled cells in the ipsilateral SVZ after ischemia. Representative pictures of BrdU-immunofluorescence staining from the rats treated with either edaravone (A–C) or control (D–F) at days 1, 4, and 7 after ischemia, respectively, being visualized with TRITC (Red). Bar⫽50 ␮m. LV, lateral ventricle.

Edaravone reduced BrdU-labeled cells in the SVZ after ischemia Cell proliferation was evaluated by BrdU incorporation. Only dispersed BrdU-positive cells were observed in the SVZ of the sham-operated group (Fig. 1C). More BrdUlabeled cells were observed in the SVZ after ischemia (Fig. 2A–F). However, cell proliferation was more prominent in the ischemic ipsilateral SVZ of the control group (Fig. 2D–F). Quantitative analysis showed that the number of BrdU-positive cells in the ischemic ipsilateral SVZ was significantly lower in the edaravone group than in the con-

trol group at one day post-ischemia (P⬍0.01; Fig. 3B). Reduction in SVZ cell proliferation was 50% and 42% in the rats that received edaravone vs. normal saline at 4 and 7 days, respectively (P⬍0.05; Fig. 3B). Edaravone decreased neural progenitor cells in the SVZ after ischemia It is known that cerebral ischemia enhances neurogenesis in the SVZ (Zhang et al., 2008). We used DCX as a marker for neural progenitor cells (Couillard-Despres et al., 2005). Few DCX-positive cells were observed in the

Fig. 3. Changes in neurological deficits and neurogenesis. (A) Effect of edaravone on neurological function evaluated by modified neurological severity score. Values are mean⫾SD. n⫽5 per time point. (B, C) Number of BrdU (B) or DCX (C)-positive cells in the ischemic ipsilateral SVZ. Values are mean⫾SD. n⫽5 per time point. * P⬍0.05 compared with control. ** P⬍0.01 compared with control. # P⬍0.01 compared with the sham.

P. Zhang et al. / Neuroscience 201 (2012) 297–306

301

Fig. 4. Changes in neural progenitor cells in the ipsilateral SVZ after ischemia. Representative pictures of DCX immunohistochemical staining from the rats treated with either edaravone (A–C) or control (D–F) 1, 4, and 7 d after ischemia, respectively, being visualized with diaminobenzidine (brown). Insets show the high magnification of regions indicated by arrows. Bar⫽150 ␮m. LV, lateral ventricle.

SVZ of the sham-operated group (Fig. 1D). More DCXpositive cells were found in the ischemic ipsilateral SVZ of the control group (Fig. 4D–F). However, DCX-positive cells were interspersed in the ischemic ipsilateral SVZ of the edaravone group (Fig. 4A–C). The number of DCXpositive cells in the ischemic ipsilateral SVZ was significantly lower in the edaravone group than in the control group at 1, 4, and 7 days post-ischemia, respectively (P⬍0.05; Fig. 3C). To confirm that DCX-positive cells were derived from newly generated cells, the sections were double-labeled with BrdU and DCX antibodies. Around two-thirds of the cells (edaravone group: 64⫾4% of BrdU-positive cells; control group: 62⫾3% of BrdUpositive cells; P⬎0.05 vs. control) were found to be DCX-BrdU immunoreactive, indicating enhanced SVZ neurogenesis after injury. A representative image of the SVZ 7 days post-ischemia is shown in Fig. 5. Edaravone repressed HIF-1␣ and VEGF protein expressions in the SVZ after injury Hypoxia-induced HIF-1␣ expression promotes NSC proliferation. ROS regulates the HIF-1 signaling pathway depending on the cell types (Koshikawa et al., 2009). VEGF is one of the growth factors regulated by HIF-1␣ and plays an essential role in the neurogenesis of NSCs. To confirm that ROS and HIF-1 signaling pathway are involved in the inhibition of neurogenesis induced by

edaravone treatment, we determined HIF-1␣ and VEGF protein levels by Western blot analysis. In the shamoperated group, both HIF-1␣ and VEGF proteins in the SVZ were detected at a relatively low level (Fig. 6A, B). After ischemia, the protein expressions of both HIF-1␣ and VEGF peaked at 6 h after ischemia and decreased thereafter (Fig. 6C, D). However, both HIF-1␣ and VEGF protein levels in the ischemic ipsilateral SVZ were significantly lower in the edaravone group compared with the control group at all studied time points, respectively (P⬍0.05; Fig. 6C, D). Edaravone decreased ROS generation in the SVZ after injury Red fluorescence appeared in the SVZ of the sham-operated group, which indicated that ROS exists in the normal brain tissue (Fig. 1E). The intensity of red fluorescence, indicating ROS production, in the ischemic ipsilateral SVZ was notable in the control group 6, 12, and 24 h after ischemia (Fig. 7D–F). Semi-quantitative analysis revealed that the ratio of fluorescence intensity was 157.5⫾47.5%, 244.5⫾25.7%, and 444.5⫾149.8% of the contralateral SVZ at 6, 12, and 24 h, respectively. When treated with edaravone, the intensity of red fluorescence in the ischemic ipsilateral SVZ became weaker at 6, 12, and 24 h after ischemia compared with the control group (Fig. 7A–

302

P. Zhang et al. / Neuroscience 201 (2012) 297–306

Fig. 5. DCX-positive cells were derived from newly generated cells. Representative pictures of BrdU-DCX double immunofluorescence staining from the rats treated with either edaravone (A–C) or control (D–F) 7 d after ischemia, being viewed under laser confocal microscopy. Most of the DCX-positive cells incorporated with BrdU. Insets show the high magnification of cells indicated by arrows. Bar⫽50 ␮m. LV, lateral ventricle.

C). The ratio was 57.0⫾31.8%, 132.3⫾16.2%, and 201.0⫾ 67.2% at 6, 12, and 24 h, respectively. As a result, treatment with edaravone significantly lowered ROS generation after ischemia (P⬍0.05).

DISCUSSION In this study, we evaluated the in vivo effects of edaravone in a rat model of focal cerebral ischemia/reperfusion injury.

Fig. 6. Changes in HIF-1␣ and VEGF protein levels in ischemic ipsilateral SVZ. (A, B) Representative pictures of HIF-1␣ (A) and VEGF (B) expressions from rats treated with either edaravone or control 6, 12, and 24 h after ischemia. (C, D) Quantitative analysis of HIF-1␣ (C) and VEGF (D) proteins at each time point. n⫽5 per time point. Values are presented as mean⫾SD. * P⬍0.05 compared with the control. # P⬍0.05 compared with the sham.

P. Zhang et al. / Neuroscience 201 (2012) 297–306

303

Fig. 7. Detection of ROS by DHE staining. Representative pictures of DHE staining from the rats treated with either edaravone (A–C) or control (D–F) at 6, 12, and 24 h after ischemia, respectively, being viewed under fluorescence microscopy. The red fluorescence was notable in the control group. Bar⫽80 ␮m. LV, lateral ventricle.

The results indicated that edaravone significantly reduced the infarct size and improved neurological scores. Interestingly, edaravone inhibited neurogenesis in the ischemic ipsilateral SVZ. Furthermore, edaravone decreased ROS generation and HIF-1␣ as well as VEGF protein expression levels within the ischemic ipsilateral SVZ. These findings suggest that edaravone may decrease SVZ neurogenesis through repressing ROS and HIF-1␣ signaling after ischemia-reperfusion injury. Several studies have reported the neuroprotective role of edaravone in experimental brain injury models. In rat transient or permanent cerebral ischemia model, edaravone prevented cortical edema, reduced the infarct volume, and improved neurological deficits (Shichinohe et al., 2004; Wu et al., 2006). Moreover, edaravone has been shown to attenuate ischemic or traumatic brain injury in adult and immature animal models (Yamamoto et al., 2009; Itoh et al., 2010). Edaravone displayed neuroprotective effects in patients with acute cerebral infarction (Watanabe et al., 2008). Our data showed a similar reduction in the infarct area and neurological severity score, consistent with the above mentioned reports, indicating that our injection protocol of edaravone protects against cerebral ischemia-reperfusion injury. The adult SVZ harbors neural stem/progenitor cells (NSPCs) (Doetsch et al., 1999; Romanko et al., 2004). The majority of cells in the adult SVZ are migrating neuroblasts (type A cells, expressing DCX) that continue to proliferate (García-Verdugo et al., 1998; Couillard-

Despres et al., 2005). Substantial evidence supports the occurrence of enhanced neurogenesis in the ipsilateral SVZ after ischemia in adult rats (Young et al., 2011; Zhang et al., 2004, 2008). Retinoid (Plane et al., 2008), bone morphogenetic protein (Chou et al., 2006), tumor necrosis factor-alpha (TNF-␣) (Iosif et al., 2008), VEGF, transforming growth factor-beta (Sun et al., 2010), and sonic hedgehog (Sims et al., 2009) signaling pathways appear important for stroke-induced SVZ neurogenesis. One recent study demonstrated that endogenous ROS and nitric oxide were essential for the proliferation of embryonic NSPCs in vitro (Yoneyama et al., 2010). In our study, we found that treatment with edaravone markedly decreased BrdU-positive cells and DCX-positive cells in the ipsilateral SVZ of rats after cerebral ischemia-reperfusion injury. Additionally, smaller infarct sizes were observed in the edaravone-treated rats. Whether neurogenesis is reduced with less severe injury is unknown (Tanaka et al., 2010). A recent study revealed that minocycline, a microglia activation inhibitor, reduced neurogenesis but did not change infarct size and behavioral function after transient cerebral ischemia in rat (Kim et al., 2009). Our findings indicate that ROS is involved in regulation of neurogenesis after stroke. Further analysis is needed to determine the relationship between infarct size and neurogenesis. HIF-1a is recognized as a multifunctional protein that regulates the expression and activity of multiple genes involved in angiogenesis, glucose metabolism, prolifera-

304

P. Zhang et al. / Neuroscience 201 (2012) 297–306

tion and self-renewal, apoptosis, and migration through both transcriptional and non-transcriptional signaling pathways (Harms et al., 2010). Overexpression of HIF-1␣ induces proliferation of NSPCs under normal oxygen conditions and under hypoxia. In contrast, knockdown of HIF-1␣ inhibited proliferation of NSPCs induced by hypoxia (Zhang et al., 2006 –2007). NSCs express HIF-1␣ and its downstream components such as VEGF. To explore the potential mechanism by which edaravone inhibits strokeinduced SVZ neurogenesis, we detected the changes in HIF-1␣ and VEGF in the ischemic ipsilateral SVZ 6, 12, and 24 h after ischemia. The results showed that treatment with edaravone attenuated the rise in HIF-1␣ and VEGF protein levels due to injury in the ischemic ipsilateral SVZ, which indicated that such treatment decreases neurogenesis by repressing HIF-1␣ signaling. ROS play a critical role in HIF-1␣ expression. ROS originating from the mitochondria has been attributed to triggering an increase in HIF-1␣ in the human hepatocellular carcinoma cell line, HepG2 (Fandrey et al., 1997), Hep3B cells (Chandel et al., 1998), cardiomyocytes (Duranteau et al., 1998), endothelial cells (Grishko et al., 2001; Pearlstein et al., 2002), and renal cells (Turcotte et al., 2003). Liu et al. reported that ROS increased HIF-1␣ and its downstream gene EPO expression in neuronal cultures after hypoxia (Liu et al., 2005). HIF-1␣ accumulation was observed in both old and young rat cerebral cortexes exposed to intermittent hypoxia, which increases ROS production (Rapino et al., 2005). Manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride, a potent scavenger of superoxide, not only prevented chronic intermittent hypoxia-induced increases in ROS, but also chronic intermittent hypoxia-evoked HIF-1␣ up-regulation in mice (Peng et al., 2006). Despite many studies of the effects of ROS on HIF-1␣ protein levels in various cell types (Koshikawa et al., 2009), it is not known whether ROS regulates HIF-1␣ in SVZ cells. In this study, treatment with edaravone decreased the levels of ROS and HIF-1␣ protein in the ischemic ipsilateral SVZ. Meanwhile, VEGF protein, a downstream component of HIF-1␣ was also reduced by edaravone treatment. Ishikawa et al. reported that edaravone inhibited the expression of HIF-1␣ and VEGF in human astrocytes exposed to hypoxia (Ishikawa et al., 2007). Overexpression of VEGF has been shown to enhance neurogenesis in the SVZ of the adult rat after a transient middle cerebral artery occlusion (Wang et al., 2007; Li et al., 2009). Considering the contribution of HIF-1␣ and VEGF to neurogenesis, our findings indicate that edaravone, via repressing ROS and HIF-1␣ signaling pathway, inhibits neurogenesis in the ipsilateral SVZ after cerebral ischemia-reperfusion injury. Further studies will be required to elucidate the upstream and downstream molecules of HIF-1␣ by which edaravone decreases neurogenesis after ischemia.

CONCLUSIONS For the first time, we have shown that edaravone decreases neurogenesis, depresses ROS generation and

HIF-1␣ as well as VEGF protein levels in the ipsilateral SVZ after focal ischemia-reperfusion, indicating that edaravone, via repressing HIF-1␣ signaling pathway, inhibits neurogenesis after stroke. Our data provide an experimental basis for understanding the contribution of ROS and HIF-1␣ signaling to neurogenesis after cerebral ischemia. This may be important for the use of free radical scavengers in the treatment of cerebral ischemia and reperfusion injury. Acknowledgments—This work was supported by National Natural Science Foundation of China (No. 30772081; 81071071) and Program for New Century Excellent Talents in University (NCET08-0436).

REFERENCES Androutsellis-Theotokis A, Leker RR, Soldner F, Hoeppner DJ, Ravin R, Poser SW, Rueger MA, Bae SK, Kittappa R, McKay RD (2006) Notch signaling regulates stem cell numbers in vitro and in vivo. Nature 442:823– 826. Bernier PJ, Vinet J, Cossette M, Parent A (2000) Characterization of the subventricular zone of the adult human brain: evidence for the involvement of Bcl-2. Neurosci Res 37:67–78. Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, Schumacker PT (1998) Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci U S A 95:11715–11720. Chou J, Harvey BK, Chang CF, Shen H, Morales M, Wang Y (2006) Neuroregenerative effects of BMP7 after stroke in rats. J Neurol Sci 240:21–29. Couillard-Despres S, Winner B, Schaubeck S, Aigner R, Vroemen M, Weidner N, Bogdahn U, Winkler J, Kuhn HG, Aigner L (2005) Doublecortin expression levels in adult brain reflect neurogenesis. Eur J Neurosci 21:1–14. Doetsch F, Caillé I, Lim DA, García-Verdugo JM, Alvarez-Buylla A (1999) Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97:703–716. Duranteau J, Chandel NS, Kulisz A, Shao Z, Schumacker PT (1998) Intracellular signaling by reactive oxygen species during hypoxia in cardiomyocytes. J Biol Chem 273:11619 –11624. Emsley JG, Mitchell BD, Kempermann G, Macklis JD (2005) Adult neurogenesis and repair of the adult CNS with neural progenitors, precursors, and stem cells. Prog Neurobiol 75:321–341. Fandrey J, Frede S, Ehleben W, Porwol T, Acker H, Jelkmann W (1997) Cobalt chloride and desferrioxamine antagonize the inhibition of erythropoietin production by reactive oxygen species. Kidney Int 51:492– 496. García-Verdugo JM, Doetsch F, Wichterle H, Lim DA, Alvarez-Buylla A (1998) Architecture and cell types of the adult subventricular zone: in search of the stem cells. J Neurobiol 36:234 –248. Grishko V, Solomon M, Breit JF, Killilea DW, Ledoux SP, Wilson GL, Gillespie MN (2001) Hypoxia promotes oxidative base modifications in the pulmonary artery endothelial cell VEGF gene. FASEB J 15:1267–1269. Harms KM, Li L, Cunningham LA (2010) Murine neural stem/progenitor cells protect neurons against ischemia by HIF-1alpha-regulated VEGF signaling. PLoS One 5:e9767. Hsieh CL, Cheng CY, Tsai TH, Lin IH, Liu CH, Chiang SY, Lin JG, Lao CJ, Tang NY (2006) Paeonol reduced cerebral infarction involving the superoxide anion and microglia activation in ischemia-reperfusion injured rats. J Ethnopharmacol 106:208 –215. Hwang KY, Oh YT, Yoon H, Lee J, Kim H, Choe W, Kang I (2008) Baicalein suppresses hypoxia-induced HIF-1alpha protein accumulation and activation through inhibition of reactive oxygen species and PI 3-kinase/Akt pathway in BV2 murine microglial cells. Neurosci Lett 444:264 –269.

P. Zhang et al. / Neuroscience 201 (2012) 297–306 Im SH, Yu JH, Park ES, Lee JE, Kim HO, Park KI, Kim GW, Park CI, Cho SR (2010) Induction of striatal neurogenesis enhances functional recovery in an adult animal model of neonatal hypoxicischemic brain injury. Neuroscience 169:259 –268. Iosif RE, Ahlenius H, Ekdahl CT, Darsalia V, Thored P, Jovinge S, Kokaia Z, Lindvall O (2008) Suppression of stroke-induced progenitor proliferation in adult subventricular zone by tumor necrosis factor receptor 1. J Cereb Blood Flow Metab 28:1574 –1587. Ishikawa A, Yoshida H, Metoki N, Toki T, Imaizumi T, Matsumiya T, Yamashita K, Taima K, Satoh K (2007) Edaravone inhibits the expression of vascular endothelial growth factor in human astrocytes exposed to hypoxia. Neurosci Res 59:406 – 412. Itoh T, Satou T, Nishida S, Tsubaki M, Imano M, Hashimoto S, Ito H (2010) Edaravone protects against apoptotic neuronal cell death and improves cerebral function after traumatic brain injury in rats. Neurochem Res 35:348 –355. Kim BJ, Kim MJ, Park JM, Lee SH, Kim YJ, Ryu S, Kim YH, Yoon BW (2009) Reduced neurogenesis after suppressed inflammation by minocycline in transient cerebral ischemia in rat. J Neurol Sci 279:70 –75. Kornack DR, Rakic P (2001) The generation, migration, and differentiation of olfactory neurons in the adult primate brain. Proc Natl Acad Sci U S A 98:4752– 4757. Koshikawa N, Hayashi J, Nakagawara A, Takenaga K (2009) Reactive oxygen species-generating mitochondrial DNA mutation up-regulates hypoxia-inducible factor-1alpha gene transcription via phosphatidylinositol 3-kinase-Akt/protein kinase C/histone deacetylase pathway. J Biol Chem 284:33185–33194. Lee SH, Kim YJ, Lee KM, Ryu S, Yoon BW (2007) Ischemic preconditioning enhances neurogenesis in the subventricular zone. Neuroscience 146:1020 –1031. Li SF, Sun YB, Meng QH, Li SR, Yao WC, Hu GJ, Li ZJ, Wang RZ (2009) Recombinant adeno-associated virus serotype 1-vascular endothelial growth factor promotes neurogenesis and neuromigration in the subventricular zone and rescues neuronal function in ischemic rats. Neurosurgery 65:771–779. Liu J, Narasimhan P, Yu F, Chan PH (2005) Neuroprotection by hypoxic preconditioning involves oxidative stress-mediated expression of hypoxia-inducible factor and erythropoietin. Stroke 36:1264 –1269. Ohira K (2011) Injury-induced neurogenesis in the mammalian forebrain. Cell Mol Life Sci 68:1645–1656. Panchision DM (2009) The role of oxygen in regulating neural stem cells in development and disease. J Cell Physiol 220:562–568. Pearlstein DP, Ali MH, Mungai PT, Hynes KL, Gewertz BL, Schumacker PT (2002) Role of mitochondrial oxidant generation in endothelial cell responses to hypoxia. Arterioscler Thromb Vasc Biol 22:566 –573. Peng YJ, Yuan G, Ramakrishnan D, Sharma SD, Bosch-Marce M, Kumar GK, Semenza GL, Prabhakar NR (2006) Heterozygous HIF-1␣ deficiency impairs carotid body-mediated systemic responses and reactive oxygen species generation in mice exposed to intermittent hypoxia. J Physiol 577:705–716. Plane JM, Whitney JT, Schallert T, Parent JM (2008) Retinoic acid and environmental enrichment alter subventricular zone and striatal neurogenesis after stroke. Exp Neurol 214:125–134. Rapino C, Bianchi G, Di Giulio C, Centurione L, Cacchio M, Antonucci A, Cataldi A (2005) HIF-1␣ cytoplasmic accumulation is associated with cell death in old rat cerebral cortex exposed to intermittent hypoxia. Aging Cell 4:177–185. Romanko MJ, Rola R, Fike JR, Szele FG, Dizon ML, Felling RJ, Brazel CY, Levison SW (2004) Roles of the mammalian subventricular zone in cell replacement after brain injury. Prog Neurobiol 74:77–99. Schänzer A, Wachs FP, Wilhelm D, Acker T, Cooper-Kuhn C, Beck H, Winkler J, Aigner L, Plate KH, Kuhn HG (2004) Direct stimulation of adult neural stem cells in vitro and neurogenesis in vivo by vascular endothelial growth factor. Brain Pathol 14:237–248.

305

Schmidt W, Reymann KG (2002) Proliferating cells differentiate into neurons in the hippocampal CA1 region of gerbils after global cerebral ischemia. Neurosci Lett 334:153–156. Schölzke MN, Schwaninger M (2007) Transcriptional regulation of neurogenesis: potential mechanisms in cerebral ischemia. J Mol Med 85:577–588. Shi Q, Zhang P, Zhang J, Chen X, Lu H, Tian Y, Parker TL, Liu Y (2009) Adenovirus-mediated brain-derived neurotrophic factor expression regulated by hypoxia response element protects brain from injury of transient middle cerebral artery occlusion in mice. Neurosci Lett 465:220 –225. Shichinohe H, Kuroda S, Yasuda H, Ishikawa T, Iwai M, Horiuchi M, Iwasaki Y (2004) Neuroprotective effects of the free radical scavenger Edaravone (MCI-186) in mice permanent focal brain ischemia. Brain Res 1029:200 –206. Sims JR, Lee SW, Topalkara K, Qiu J, Xu J, Zhou Z, Moskowitz MA (2009) Sonic hedgehog regulates ischemia/hypoxia-induced neural progenitor proliferation. Stroke 40:3618 –3626. Sun J, Zhou W, Sha B, Yang Y (2010) Ischemia induced neural stem cell proliferation and differentiation in neonatal rat involved vascular endothelial growth factor and transforming growth factor-beta pathways. Brain Dev 32:191–200. Tanaka Y, Tanaka R, Liu M, Hattori N, Urabe T (2010) Cilostazol attenuates ischemic brain injury and enhances neurogenesis in the subventricular zone of adult mice after transient focal cerebral ischemia. Neuroscience 171:1367–1376. Tang B, Qu Y, Wang D, Mu D (2010) Targeting hypoxia inducible factor-1␣: a novel mechanism of ginsenoside rg1 for brain repair after hypoxia/ischemia brain damage. CNS Neurol Disord Drug Targets 10:235–238. Turcotte S, Desrosiers RR, Béliveau R (2003) HIF-1␣ mRNA and protein upregulation involves Rho GTPase expression during hypoxia in renal cell carcinoma. J Cell Sci 116:2247–2260. Wang YQ, Guo X, Qiu MH, Feng XY, Sun FY (2007) VEGF overexpression enhances striatal neurogenesis in brain of adult rat after a transient middle cerebral artery occlusion. J Neurosci Res 85:73– 82. Watanabe T, Tahara M, Todo S (2008) The novel antioxidant edaravone: from bench to bedside. Cardiovasc Ther 26:101–114. Wu T, Ding XS, Wang W, Wu J (2006) MCI-186 (3-methyl-1-phenyl-2pyrazolin-5-one) attenuated simulated ischemia/reperfusion injury in cultured rat hippocampal cells. Biol Pharm Bull 29:1613–1617. Yamamoto Y, Yanagisawa M, Tak NW, Watanabe K, Takahashi C, Fujisawa A, Kashiba M, Tanaka M (2009) Repeated edaravone treatment reduces oxidative cell damage in rat brain induced by middle cerebral artery occlusion. Redox Rep 14:251–258. Yoneyama M, Kawada K, Gotoh Y, Shiba T, Ogita K (2010) Endogenous reactive oxygen species are essential for proliferation of neural stem/progenitor cells. Neurochem Int 56:740 – 746. Young CC, Brooks KJ, Buchan AM, Szele FG (2011) Cellular and molecular determinants of stroke-induced changes in subventricular zone cell migration. Antioxid Redox Signal 14:1877– 1888. Zhang CP, Zhu LL, Zhao T, Zhao H, Huang X, Ma X, Wang H, Fan M (2006 –2007) Characteristics of neural stem cells expanded in lowered oxygen and the potential role of hypoxia-inducible factor1alpha. Neurosignals 15:259 –265. Zhang H, Vutskits L, Pepper MS, Kiss JZ (2003) VEGF is a chemoattractant for FGF-2-stimulated neural progenitors. J Cell Biol 163:1375–1384. Zhang P, Li J, Liu Y, Chen X, Kang Q, Zhao J, Li W (2009) Human neural stem cell transplantation attenuates apoptosis and improves neurological functions after cerebral ischemia in rats. Acta Anaesthesiol Scand 53:1184 –1191. Zhang P, Li J, Liu Y, Chen X, Lu H, Kang Q, Li W, Gao M (2011) Human embryonic neural stem cell transplantation increases subventricular zone cell proliferation and promotes peri-infarct

306

P. Zhang et al. / Neuroscience 201 (2012) 297–306

angiogenesis after focal cerebral ischemia. Neuropathology 31:384 –391. Zhang R, Zhang Z, Wang L, Wang Y, Gousev A, Zhang L, Ho KL, Morshead C, Chopp M (2004) Activated neural stem cells contribute to strokeinduced neurogenesis and neuroblast migration toward the infarct boundary in adult rats. J Cereb Blood Flow Metab 24:441–448.

Zhang RL, Zhang ZG, Chopp M (2008) Ischemic stroke and neurogenesis in the subventricular zone. Neuropharmacology 55:345–352. Zhang RL, Zhang ZG, Zhang L, Chopp M (2001) Proliferation and differentiation of progenitor cells in the cortex and the subventricular zone in the adult rat after focal cerebral ischemia. Neuroscience 105:33– 41.

(Accepted 2 November 2011) (Available online 15 November 2011)