Temporal activation of Nrf2 in the penumbra and Nrf2 activator-mediated neuroprotection in ischemia–reperfusion injury

Free Radical Biology and Medicine 72 (2014) 124–133

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Original Contribution

Temporal activation of Nrf2 in the penumbra and Nrf2 activator-mediated neuroprotection in ischemia–reperfusion injury Toshinori Takagi a,b, Akira Kitashoji a, Takao Iwawaki c, Kazuhiro Tsuruma a, Masamitsu Shimazawa a, Shinichi Yoshimura b,1, Toru Iwama b, Hideaki Hara a,n a

Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, Gifu 501-1196, Japan Department of Neurosurgery, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan c Advanced Scientific Research Leaders Development Unit, Gunma University, Maebashi City, Gunma 371-8511, Japan b

art ic l e i nf o

a b s t r a c t

Article history: Received 19 February 2014 Received in revised form 3 April 2014 Accepted 4 April 2014 Available online 15 April 2014

Oxidative stress plays a critical role in mediating tissue injury and neuron death during ischemia– reperfusion injury (IRI). The Keap1–Nrf2 defense pathway serves as a master regulator of endogenous antioxidant defense, and Nrf2 has been attracting attention as a target for the treatment of IRI. In this study, we evaluated Nrf2 expression in IRI using OKD (Keap1-dependent oxidative stress detector) mice and investigated the neuroprotective ability of an Nrf2 activator. We demonstrated temporal changes in Nrf2 expression in the same mice with luciferase assays and an Nrf2 activity time course using Western blotting. We also visualized Nrf2 expression in the ischemic penumbra and investigated Nrf2 expression in mice and humans using immunohistochemistry. Endogenous Nrf2 upregulation was not detected early in IRI, but expression peaked 24 h after ischemia. Nrf2 expression was mainly detected in the penumbra, and it was found in neurons and astrocytes in both mice and humans. Intravenous administration of the Nrf2 activator bardoxolone methyl (BARD) resulted in earlier upregulation of Nrf2 and heme oxygenase-1. Furthermore, BARD decreased infarction volume and improved neurological symptoms after IRI. These findings indicate that earlier Nrf2 activation protects neurons, possibly via effects on astrocytes. & 2014 Elsevier Inc. All rights reserved.

Keywords: Bardoxolone methyl Ischemia–reperfusion injury Neuroprotection Nrf2 Oxidative stress Free radicals

Stroke is a common cause of death and the leading cause of adult disability worldwide [1]. Recanalization after cerebral ischemia is the most effective method for treating acute cerebral ischemia and correcting hypoxia. Recombinant tissue-plasminogen activator can be used only within 4.5 h of stroke onset [2], and mechanical thrombectomy with stent retrievers is approved within 8 h of onset [3]. Successful recanalization rates have increased with medical advancements, but clinical outcomes have not been sufficiently improved [3]. Paradoxically, reperfusion from recanalized cerebral vessels can promote ischemia–reperfusion injury (IRI), which is a major medical problem that requires additional investigation [4,5]. There are a variety of IRI-induced cascade reactions, including calcium imbalance, excitotoxicity, and mitochondrial dysfunction [6]. In addition, increasing evidence suggests a critical role for oxidative stress in mediating tissue injury and neuron death during IRI [7]. Free radical production after IRI is enhanced owing to elimination of endogenous antioxidative systems in ischemic tissues, especially after reperfusion [8]. Elevated levels of reactive oxygen n

Corresponding author. Fax: þ81 58 230 8126. E-mail address: [email protected] (H. Hara). 1 Present address: Department of Neurosurgery, Hyogo College of Medicine, Nishinomiya City, Hyogo 663-8501, Japan. http://dx.doi.org/10.1016/j.freeradbiomed.2014.04.009 0891-5849/& 2014 Elsevier Inc. All rights reserved.

species (ROS) can directly disrupt the structures of lipids, proteins, and DNA and induce cell death in various ways [9]. Furthermore, ROS can serve as intracellular signaling molecules and control inflammation or cellular injuries [10]. The redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) plays a key role in orchestrating cellular antioxidant defenses and maintaining redox homeostasis [11,12]; it also regulates the expression of numerous ROSdetoxifying and antioxidant genes (phase II genes) [11,13]. Under normal conditions, Nrf2 interacts with Kelch-like ECH-associated protein 1 (Keap1), which limits Nrf2-mediated gene expression [14,15]. Upon activation, the Keap1–Nrf2 complex is dissociated, and Nrf2 translocates into the nuclei to bind antioxidant-response elements (AREs) and activate ARE-dependent transcription of phase II and antioxidant defense enzymes, including heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1, to attenuate cellular oxidative stress [16–18]. HO-1 is a ubiquitous and redox-sensitive inducible stress protein that can exert a potent indirect antioxidative function by degrading heme to CO, iron, and biliverdin [19]. Recently, many studies have shown that pharmacological activation of Nrf2 leads to neuroprotection in IRI by upregulating AREs [5,20–22]. Among them, bardoxolone methyl (BARD) is an

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attractive candidate drug for clinical application, and it has been tested in a phase II study of patients with chronic kidney disease and type 2 diabetes [23]. However, whether Nrf2 is expressed or activated in the acute ischemic phase within the penumbra and/or core of the acute ischemic brain remains controversial [24,25]. Hence, we employed a Keap1-dependent oxidative stress detector–luciferase (OKD-LUC) mouse model to visualize Nrf2 expression from the onset of brain ischemia through the early hours after reperfusion and investigated Nrf2 localization using a mouse model with green fluorescent protein (GFP)-labeled Nrf2 (OKD-V) [34]. We also examined cell-type-specific Nrf2 expression in coronal brain sections of OKD-V mice [26]. Similar immunohistochemistry (IHC) experiments were performed in human tissue samples. Moreover, we investigated the neuroprotective effect of intravenous administration of the Nrf2 activator BARD after Nrf2 and HO-1 activation.

Materials and methods Animals All animal protocols were conducted in accordance with the ARRIVE (Animal Research: Reporting in vivo Experiments) guidelines and were approved by the animal experimentation committees of Gifu Pharmaceutical University, Japan. All wild-type mice (ddY mice) were purchased from Japan SLC Ltd. (Hamamatsu, Japan). Animals were housed at 2472 1C under a 12-h light–dark cycle. Food and water were available ad libitum. OKD-V and OKD-LUC mice To detect oxidative stress via the Keap1–Nrf2 system, we used OKD-V and OKD-LUC mice, which were provided by Dr. Takao

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Iwawaki from Gunma University [26]. All mice were on a C57BL/6 background. The design and construction of OKD-V and OKD-LUC mice are shown in Fig. 1. p(3  ARE)TKbasal-hNrf2(1–433)GL4-FLAG was generated as the OKD-V construct. The OKD-V construct has a 3  ARE promoter (blue), human Nrf2 (amino acids 1–433, blue gradient), and FLAG-tagged GFP (GL4, green). Under normal conditions, transcription of the OKD construct is not induced, and the leaked OKD protein is degraded by the Keap1 (orange) system. Upon oxidative stress, the OKD construct is transcriptionally induced by the 3  ARE, and the resulting protein is stabilized by the Keap1 system. Thus, we detected fluorescence only in cells experiencing oxidative stress. We also employed OKD-LUC mice, the construction of which was almost the same as that of OKD-V mice, except that luciferase was used in place of GFP. The transgenic offspring were screened by polymerase chain reaction (PCR) using the primers 50 -ATCACCAGAACACTCAGTGG-30 and 50 -ACTCGGCGTAGGTAATGTCC-30 , for OKD-LUC mice, and 50 ATCACCAGAACACTCAGTGG-30 and 50 -TCGTGCTGCTTCATGTGGTC30 for OKD-V mice (Fig. 1). Mouse focal brain ischemia model Male mice ages 8–12 weeks were used for focal brain ischemia experiments. There was no significant difference in weight and age between any of the transgenic and wild-type groups. The operators were blinded to the treatment status of the animals in all experiments. We employed a previously described transient middle cerebral artery occlusion (MCAO) model induced by means of an intraluminal suture [27]. The mice were anesthetized with 2.0 to 2.5% isoflurane (Mylan, Inc., Canonsburg, PA, USA) and maintained using 1.0 to 1.5% isoflurane in 70% N2O/30% O2, delivered via a face mask with an animal general anesthesia

Fig. 1. Schematic representation of the OKD construct. (A) Human Nrf2 contains a Keap1 binding site (Neh2) and a DNA binding site (Neh1). The OKD construct includes a 3  ARE promoter (arrow), partial human Nrf2 (1–433), and FLAG-tagged GFP or luciferase. (B) Under oxidative stress, transcription of the OKD construct is induced, and OKD protein is stabilized by cancellation of Keap1-mediated degradation. (C) The OKD construct is detectable by PCR only in transgenic mice. (D) Under normal conditions, OKD construct transcription is not induced, and the OKD protein is degraded by the Keap1 system.

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machine (Soft Lander, Sin-ei Industry Co., Ltd., Saitama, Japan). In each mouse, regional cerebral blood flow was monitored by laserDoppler flowmetry (Omega-Flow flo-N1; Omegawave, Inc., Tokyo, Japan) except for luciferase assay studies. A flexible probe was fixed to the skull (2-mm posterior and 6-mm lateral to bregma). We excluded mice that did not demonstrate a significant reduction (to o40% baseline relative cerebral blood flow (rCBF) values) in CBF. In luciferase assays, CBF was monitored by laser speckle flowgraphy (LSFG-ANW; Softcare Ltd., Fukuoka, Japan). Two hours after MCAO, the brain was reperfused by removal of the intraluminal suture. Body temperature of all animals was maintained at 37.0 to 37.5 1C with the aid of a heating pad and heating lamp throughout these procedures. After the surgery, the mice were housed under the preoperative conditions until further experimentation. We used a total of 114 mice (ddY 54, OKD-LUC 24, OKD-V 36) in this study and excluded 11 mice (ddY 4, OKD-LUC 4, OKD-V 3) based on the following exclusion criteria that were set before the experiments: animals that died from excessive anesthesia or procedural problems that occurred during operations and those that died up to sampling after operations. Furthermore, as failures of MCAO/reperfusion, animals that did not demonstrate a significant reduction (to o40% baseline rCBF values) in CBF and animals in which the nylon monofilament had escaped at the time of reperfusion were excluded. Luciferase assay Bioluminescence imaging was performed as previously described [28,29]. OKD-LUC mice were imaged using the IVIS imaging system (Lumina, Caliper Life Sciences, Hopkinton, MA, USA). Twenty minutes before the imaging session, the mice received an intraperitoneal (ip) injection of the D-luciferin potassium salt solution (Promega, Madison, WI, USA). Each mouse received 150 mg/kg luciferin solution (20 mg/ml). The mice were then anesthetized with an isoflurane/oxygen gas mix, and the scalp skin was opened briefly and imaged for 2 min under constant anesthesia. Bioluminescence values were quantified from images displaying surface radiance using circular regions of interest (ROIs) and then converted to the total flux of photons (photons/s) using Living Image 4.1 software (Caliper Life Sciences). Drug treatment BARD was purchased from AdooQ Bioscience LLC (Irvine, CA, USA). The stock solution was prepared by dissolving BARD in dimethyl sulfoxide (Nacalai Tesque, Kyoto, Japan) at a concentration of 10 mM as described previously [30]. The working solution was prepared by diluting the stock solution with phosphatebuffered saline (PBS). Mice were randomly divided into BARD treatment and vehicle groups. The BARD groups were randomly treated with a total volume of 0.2 ml (2 or 0.6 mg/kg BARD) that was administered intravenously (iv) just before reperfusion. Vehicle-treated control mice were injected with the same volume of diluents. Infarct volume measurement At 22 h after reperfusion, the mice were decapitated. The brains were removed immediately and were cut to obtain 2-mm-thick slices using a mouse brain matrix (RBM-2000C, Activational Systems, Warren, MI, USA). These slices were immersed in 2,3,5triphenyltetrazolium chloride (2%; Sigma–Aldrich, St. Louis, MO, USA) for 10 min. Infarct volume was measured by an examiner blinded to treatment allocation using ImageJ software version

1.43h (National Institutes of Health (NIH), Bethesda, MD, USA), as described previously [27]. Behavioral measurements All behavioral tests were conducted in a quiet and low-light room 24 h after ischemia by an experimenter blinded to the treatment groups. Garcia test The Garcia score (3–18) was calculated by combining the scores on the following six tests: spontaneous activity, symmetry in the movement of four limbs, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch [31]. Each test had three possible points according to the neurological presentation as described previously [31]. Grid walk test The grid walk test evaluates the ability to accurately place the forepaws on the rungs of a grid during spontaneous movement as described previously [32]. The mice were placed on a grid of 0.24 mm (length, width) with 10-mm2 openings to explore freely for 2 min. Step errors were assessed and videotaped by a camera placed under the grid. We counted a step error when the limb or the foot slipped through the grid hole. The percentage step failure of the affected side was also calculated. Western blotting and immunoprecipitation Mice were deeply anesthetized and decapitated at 6, 24, or 48 h after ischemia. Brains were quickly removed, and the brains were cut into 2-mm coronal sections and separated into ipsilateral and contralateral sides and the cortex and striatum. Tissues were homogenized in 10 ml/g tissue ice-cold lysis buffer (50 mM Tris– HCl, pH 8.0, containing 150 mM NaCl, 50 mM EDTA, 1% Triton X-100, and protease/phosphatase inhibitor mixture). Immunoprecipitation was performed using a Classic IP Kit (Thermo Fisher Scientific, Waltham, MA, USA). A sample obtained from the shamcontrol or ischemic hemisphere was loaded, and the separated proteins were subsequently transferred. For immunoblotting, the following primary antibodies were used: monoclonal anti-GFP antibody (1:1000; Medical & Biological Laboratories, Nagoya, Japan) and monoclonal anti-HO-1 antibody (1:500; GeneTex, Irvine, CA, USA). Immunoreactive bands were visualized using ImmunoStar LD (Wako Pure Chemical Industries, Osaka, Japan). Band intensities were measured using a Lumino imaging analyzer (LAS-4000; Fujifilm, Tokyo, Japan). Expression level differences were analyzed with ImageJ software version 1.43h (NIH) by measuring band intensities. Histopathology and IHC Sham-operated or ischemic mice were injected with sodium pentobarbital (Nembutal, 50 mg/kg, ip; Dainippon Sumitomo Pharma, Osaka, Japan) and then perfused through the left ventricle with 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). Brains were removed after 15-min perfusion fixation at 4 1C and then immersed in the same fixative solution overnight at 4 1C. They were then immersed in 25% sucrose in 0.1 M PB for 24 h and finally frozen in liquid nitrogen. Coronal sections were cut on a cryostat at 20 1C and stored at 80 1C until use. The sections were stained for Nissl substance with 0.1% thionin, dehydrated by passage through an ascending ethanol series,

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cleared in xylene, and coverslipped with Euikit (O. Kindler, Freiburg, Germany). For IHC with the chromogen diaminobenzidine (DAB), the sections were treated with Tris-buffered saline (TBS)/0.3% Triton X-100 containing 0.3% H2O2 for 3 h, rinsed for 1 h in the same solution, washed three times with TBS, and blocked for 30 min with 2% (w/v) Block Ace (Dainippon Sumitomo Pharma) dissolved in TBS (blocking solution). Next, the sections were incubated overnight at 4 1C with antibody against HO-1 (1:500; GeneTex). After a wash with TBS, the sections were incubated for 3 h with anti-rat IgG goat antibody (1:1000; Vector Laboratories, Burlingame, CA, USA). The sections were then incubated in ABC solution (Elite ABC; Vector Laboratories) with gentle shaking at 4 1C overnight and further treated according to the glucose oxidase–DAB– nickel method. Finally, the sections were stained with 3,30 -diaminobenzidine tetrahydrochloride (Dojindo Laboratories, Kumamoto, Japan) for 5–10 min. For IHC with fluorescence, the sections were blocked with 2% goat serum in PBS and then incubated at room temperature or 4 1C with the following primary antibodies: monoclonal anti-neuronal nuclei (NeuN) antibody (1:200; Millipore, Billerica, MA, USA) and monoclonal anti-glial fibrillary acidic protein (GFAP) antibody (1:500; Millipore). Then, they were incubated for 1 h with Alexa Fluor 546 F(ab0 )2 fragment of goat anti-mouse IgG (H1L) antibody. The sections were observed under a confocal microscope (FV10i; Olympus, Tokyo, Japan). Cell counting and data analysis To standardize the measurements, predesignated squares at a peri-infarct or region destined to infarct (based on cresyl violet staining) were counted in three randomly selected fields of coronal sections per mouse brain for each staining. ImageJ software was used to analyze each picture. These procedures were performed by an examiner blinded to the treatment. Human tissue specimens Human tissue specimens were obtained from patients who had undergone surgery at the Department of Neurosurgery in Gifu University Hospital. The use of surgical specimens for IHC was approved by the institutional review board of Gifu University (No. 25–46), and all patients or their representatives signed informed written consent forms. The stroke patient was a woman in her 60s who developed a large hemispheric infarction due to cardiogenic embolism and underwent internal and external decompression 24 h after symptom onset owing to brain herniation. The tissue specimen was from the temporal cortex. The control patient was a woman in her 20s who presented with intractable epilepsy due to a cavernous malformation in the temporal lobe and had no other medical history; her neurological status was completely normal with the exception of seizures. The patient underwent an anterior temporal lobectomy, including removal of the vascular malformation. The tissue specimen was histologically normal cortex. Statistical analysis All values are expressed as the mean 7 standard error of mean. Quantitative variables were statistically analyzed using the Student two-tailed t test for two-group comparisons, Wilcoxon signed-rank tests for nonparametric values, and one-way analyses of variance (ANOVAs) followed by Dunnett's test for multiple pairwise comparisons. p values less than 0.05 were considered statistically significant. All statistical analyses were performed using JMP 10 software (SAS Institute, Inc., Cary, NC, USA).

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Results Time course of Nrf2 expression after IRI Fig. 2 shows the real-time imaging of the Nrf2 response after transient MCAO and reperfusion, temporal CBF reduction by laser speckle, and final infarct region. In this experiment, two mice in each group were excluded because of death from excessive anesthesia. Fig. 2A depicts the temporal change in Nrf2 expression and CBF in the same mouse. In the luciferase assay Nrf2 was not observed early after ischemia and reperfusion, and Nrf2 seemed to peak at 24 h after ischemia and then decline but remain at a high level at 48 h (Fig. 2A and C). This finding indicates that endogenous activation of Nrf2 was not upregulated early in ischemia or reperfusion. CBF was significantly reduced after MCAO, and CBF reduction was recovered after reperfusion (Fig. 2A and D). In addition to the infarction area (Fig. 2E), the region of Nrf2 activation was mainly in the peri-infarct area (Fig. 2A). Time course of Nrf2 and HO-1 expression in IRI To confirm the results of the time-course study, Western blots were performed using OKD-V mice. The time course of Nrf2–GFP expression in IRI is shown in Fig. 3A. Cortex and striatum were evaluated separately. In this Western blot analysis, three mice were excluded (two died up to sampling and one did not decrease CBF enough). In the cortex, Nrf2 expression was not significantly increased 6 h after ischemia compared to the contralateral hemisphere, but it increased and peaked at 24 h and then decreased (Fig. 3A, left). In the striatum, Nrf2 expression was increased at 24 h but not at 6 or 48 h (Fig. 3A, right). Next, we evaluated the time course of HO-1 expression, a typical downstream ARE under Nrf2 control. In the cortex, HO-1 expression was increased after 24 h and continued to increase at 48 h (Fig. 3B, left). In the striatum, HO-1 expression showed the same trend as Nrf2 (Fig. 3B, right). In IRI, increased Nrf2 expression peaked at 24 h in both the cortex and the striatum. On the contralateral side, Nrf2 expression did not increase in the cortex or striatum. Association of Nrf2 expression and infarct Additional IHC experiments were performed to confirm Nrf2 expression in the penumbra. Nrf2–GFP expression and infarction in coronal sections are shown in Fig. 3C. Based on the result of cresyl violet-stained infarct and Nrf2–GFP expression 24 h after ischemia, the antioxidative stress response via Nrf2 was confirmed mainly in the penumbra and not in the ischemic core. In addition to IHC labeling, HO-1 expression was mainly observed in the penumbra in the cortex and observed in both the ischemic core and the penumbra in the striatum. On the contralateral side, Nrf2 upregulation was not observed in the cortex or striatum. Cell-type-specific Nrf2 expression To identify which cell types expressed Nrf2, immunofluorescence studies were performed for Nrf2–GFP in combination with the neuronal cell marker NeuN or the astroglial cell marker GFAP in peri-infarct regions at 24 h after 2 h MCAO. In this procedure, one mouse was excluded owing to death before sampling. Nrf2–GFP immunofluorescence was strongly visualized in the cytoplasm of brain cells. This is probably because the Nrf2–GFP fusion protein does not have the nuclear localization signaling motif(s) associated with the DNA binding domain of Nrf2 that is necessary for the nuclear localization of the fusion protein. Double-immunofluorescence studies for Nrf2–GFP and cell-type-specific markers revealed that the Nrf2 immunoreactivity was apparently colocalized with NeuN and GFAP

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Fig. 2. Visualization of Nrf2 expression in IRI using the luciferase assay. (A) in vivo imaging of Nrf2 expression before and 1, 6, 24, and 48 h after ischemia in the same mouse (top). The color calibration at the right indicates photon counts. CBF reductions by laser speckle at each time point are shown at the bottom. (B) Scheme of ROI measured by luciferase activity. (C) Plot of the data obtained by measuring luciferase activity at the ischemic lesion and on the contralateral side (photons per second). Nrf2 expression was increased significantly at 24 and 48 h after ischemia. np o 0.05 vs contralateral, Student's t test, n ¼ 4. (D) CBF detected using laser speckle. CBF was reduced by MCAO, and recovery was observed after reperfusion compared to contralateral at each time point. nnp o 0.01, np o 0.05 vs contralateral, Student's t test, n ¼ 4. (E) Analysis of infarction by TTC staining in sagittal and coronal sections.

(Fig. 4A). The number of merged cells was significantly increased in the peri-infarct area (penumbra) (Fig. 4B). In contrast, Nrf2 immunofluorescence was very faintly visualized in the contralateral side. After IRI, Nrf2 expression was observed in both neurons and astrocytes.

mouse model, and astrocytes did not activate Nrf2 under normal conditions.

Nrf2 expression in human stroke

Earlier expression of Nrf2 and HO-1 by intravenous BARD administration

Although there are many reports regarding Nrf2 neuroprotection, no study has evaluated Nrf2 activation in stroke in humans. Therefore, we assessed Nrf2 activation in human brain specimens. The resected cerebral ischemia specimens included both infarcted and peri-infarcted areas, and many neurons and glia survived throughout ischemia. The double-immunofluorescence experiments revealed that the Nrf2 immunoreactivity was apparently colocalized with NeuN and GFAP in the peri-infarct region but not in the infarction area (Fig. 4C). On the other hand, control Nrf2 colocalized with NeuN but not GFAP in the control samples. These results indicate that Nrf2 was activated in neurons and astrocytes of the peri-infarct area in both human stroke samples and the

Although endogenous Nrf2 activation was not observed in early IRI, we evaluated the treatment potential of Nrf2 activation in the mouse model. BARD was administrated intravenously 2 h after start of MCAO and just before reperfusion. Intravenous administration of BARD with or without MCAO treatment resulted in earlier Nrf2 upregulation at 1 and 4 h after injection using the luciferase assay (Fig. 5A). With MCAO treatment, Nrf2 expression was significantly increased from 1 h after reperfusion by BARD treatment compared to vehicle group, and Nrf2 expression at 4 h was increased compared to the MCAO (  ) group. After 24 h, increased Nrf2 expression did not differ between the vehicle and the BARD groups (Fig. 5A, right). Two mice were excluded in this

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Fig. 3. Time course of Nrf2–GFP and HO-1 expression as determined using Western blotting, and the relationship between infarct area and Nrf2 or HO-1 expression after IRI. (A) Time course of Nrf2–GFP expression in both the cortex and the striatum of OKD-V mice. np o 0.05 vs sham, one-way ANOVA followed by Dunnett's test. #p o 0.05 vs contralateral. Student's t test, n ¼ 5 to 7, n ¼ 4 (sham). Representative bands from the Western blotting analysis of Nrf2–GFP and β-actin are shown at the bottom. (B) Time course of HO-1 expression in the cortex and the striatum. np o 0.05 vs sham, one-way ANOVA followed by Dunnett's test. #p o 0.05 vs contralateral. Student's t test, n ¼ 5 to 7, n ¼ 4 (sham). Representative bands from the Western blotting analysis of HO-1 and β-actin are shown at the bottom. (C) Relationship between infarct area and Nrf2 or HO-1 expression. The vertical axis denotes the distance of the coronal section from the front of brain, and the horizontal axis indicates the evaluation method. Infarctions were evaluated by cresyl violet staining (first column). The antioxidative stress response via Nrf2 was evaluated by GFP signal from OKD mice (second column). Both images merged are shown in the third column. The black dotted lines show the cresyl violet-stained infarct and the merged image, and the white dotted lines show the brain section outline. The Nrf2 antioxidative stress response was mainly confirmed in the penumbra area but not in the ischemic area. HO-1 immunostaining is shown in the fourth column, and the black dotted lines indicate the area of HO-1 expression.

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Fig. 4. Immunostaining for Nrf2 and cell-type-specific markers. (A) Representative IHC staining for Nrf2 and NeuN in each infarction and peri-infarction area in the left cortex and striatum (left). Representative IHC staining for Nrf2 and GFAP (right). Scale bar, 20 μm. (B) Quantification of both Nrf2- and NeuN- or GFAP-immunoreactive cells. The number of Nrf2/NeuN-positive cells in the peri-infarct area was significantly higher: nnp o 0.01 vs infarction; Student's t test; n ¼ 3 for each group. The number of Nrf2/GFAP-positive cells in the peri-infarct area was significantly higher: nnp o 0.01, np o 0.05 vs infarction; Student's t test; n ¼ 3 for each group. (C) Nrf2 expression in human cerebral ischemia. Representative photographs showing double immunostaining for Nrf2 and NeuN or GFAP in human control brain and human stroke patient brain (n ¼ 1 for each group). Scale bar, 20 μm (overview) and 10 μm (enlarged).

experiment (one died owing to excessive anesthesia, one died before imaging). Although Nrf2 in the OKD transgenic mice did not contain DNA-binding sites, we performed an additional investigation using wild-type mice to evaluate the neuroprotective effect of BARD. In Western blot analysis, three mice were excluded (two in the BARD-treated group and one in the vehicle group). Nrf2 expression was increased by iv BARD from 1 h after injection as evaluated

by Western blot in ddY mice (Fig. 5B). In addition, HO-1, a downstream antioxidant, was upregulated 4 h after BARD injection (Fig. 5B). In addition, BARD attenuated the infarct volume and improved outcomes (Fig. 5C and D). In the infarction and neurological assessment study, two mice were excluded because of insufficient CBF reduction (one in the vehicle group and one in the 2 mg/kg BARD treatment group). For both dosages, infarct volume was decreased, and forelimb use was improved (Fig. 5C and D).

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Fig. 5. Earlier activation of Nrf2 and HO-1 and neuroprotection by BARD treatment. (A) Luciferase assay in OKD-LUC mice and earlier activation of Nrf2. Nrf2 expression was increased 1 and 4 h after BARD treatment both with and without MCAO treatment; an additional increase was detected in the MCAO treatment group at 4 h. However, there was no difference between the vehicle and the BARD-treated groups with MCAO treatment at 24 h in the right graph. np o 0.05, nnp o 0.001 vs pretreatment, Dunnett's comparison test. #p o 0.05, ##p o 0.01 vs vehicle group, Student's t test. ††p o 0.01 vs MCAO (  ) group, Student's t test, n ¼ 3 each group. (B) Nrf2 and HO-1 expression levels in wild-type mice were evaluated by Western blotting. Nrf2 was upregulated at 1 and 4 h compared with the vehicle group, and HO-1 was increased 4 h after reperfusion. np o 0.05, nnp o 0.01 vs reperfusion for 1 h, Student's t test. ##p o 0.01 vs vehicle, Student's t test, n ¼ 6 in the BARD-treated group and n ¼ 4 or 5 in the vehicle group. (C) BARD decreased the infarct volumes in both the 0.6 and the 2 mg/kg groups. np o 0.05 vs vehicle group, Student's t test, n ¼ 9 or 10. Representative infarction by TTC staining in each group is shown on the right. (D) Neurological examinations 24 h after ischemia. (Left) BARD improved forelimb use in both treatment groups. The axis bar shows the median. np o 0.05 vs vehicle, Wilcoxon signed-rank test, n ¼ 9 or 10. (Right) BARD treatment improved the total neurological symptoms assessed by the Garcia score only in the 2 mg/kg group. The axis bar shows the median Garcia score. np o 0.05 vs vehicle, Wilcoxon signed-rank test, n ¼ 9 or 10.

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However, total neurological symptoms as evaluated by the Garcia score were improved in the higher dosage group (2 mg/kg group; Fig. 5D).

Discussion IRI is still a major medical problem that requires further study [4,5], and oxidative stress plays a critical role in mediating tissue injury and neuron death during IRI [7,20,33]. The Keap1–Nrf2 defense pathway serves as a master regulator of endogenous antioxidant capability; hence, Nrf2 has been attracting attention as an IRI treatment target [11–13,34]. First, we investigated early Nrf2 expression and the location of activated Nrf2. We also assessed the time course of Nrf2 expression in both the cortex and the striatum by measuring luciferase and protein levels. Both sets of experiments indicated that Nrf2 expression reached a peak 24 h after the start of MCAO. Past reports estimated the time course of Nrf2-positive cells by immunostaining and found that Nrf2 expression was increased 4–48 h after ischemia [20,24,35]. These past findings indicated that endogenous Nrf2 upregulation was started early in IRI. This discrepancy was due to the low resolution of in vivo imaging or Western blotting, whereas that of IHC was sufficient to detect early Nrf2 expression in IRI. However, the low level of Nrf2 activation in the early phase of IRI cannot protect neurons. In fact, the administration of Nrf2 inducers pre- or postonset attenuated infarction volume and improved neurological deficits [5,21,22,35]. Therefore, it may be necessary for neuroprotection from IRI to increase Nrf2 activation to a degree that is detectable by Western blot or luciferase assay. Our findings demonstrated that Nrf2 expression was mainly in the penumbra as opposed to the ischemic core, which is in accordance with a previous study [24] that first described the precise regions of Nrf2 and HO-1 expression in IRI. Furthermore, the present results demonstrate that Nrf2 activation occurs both in a mouse model and in actual human cerebral ischemia. Because endogenous Nrf2 upregulation was not sufficient to protect neurons early in ischemia and peaked 24 h after ischemia, pharmacological activation of Nrf2 has great potential to improve

neuronal survival after ischemia. In many studies, pharmacological activation of Nrf2 was shown to protect more neurons from IRI and reduce the ischemic area [5,21]. We found that iv BARD administration at 2 h after ischemia onset increased Nrf2 and HO-1 expression levels in the early phase of reperfusion. In fact, BARD posttreatment decreased infarct volume and improved neurological symptoms. These results indicate that an Nrf2 activator is able to protect neurons against IRI. The mechanism of BARD is thought to be the dissociation of Keap1 from Nrf2 by interacting with Cys-151 of Keap1 through a Michael addition [36]. Upon synthesis, the Nrf2 protein is rapidly degraded by the 26S proteasome under normal conditions, and the half-life of Nrf2 is reported as approximately 15 min [17]. Chemical binding of specific reactive cysteine residues (Cys-151) of Keap1 disrupts Keap1-mediated Nrf2 ubiquitination and results in Nrf2 accumulation/activation [37]. As with other Nrf2 inducers, it is reasonable to activate Nrf2 by blocking its degradation. A hypothetical schematic depicting BARD-mediated neuroprotection is shown in Fig. 6. In the setting of brain ischemia, the first several hours after onset are thought to be very important in preventing neurons from undergoing cell death, as the therapeutic time window of recanalization is limited to 4.5 h [2]. Many studies have assessed pretreatment with an Nrf2 activator, but they revealed low efficacy of postonset treatment [5,21]. Therefore, it is meaningful that we observed earlier upregulation of Nrf2 and HO-1 within 4 h and a neuroprotective effect of Nrf2 activator administered after ischemia onset. A study of 2-cyano-3,12-dioxooleana-1,9 dien-28-oyl imidazoline revealed that low-dose triterpenoid attenuated ischemic neuronal injury when applied after ischemia onset [22]. We found that Nrf2 expression was significantly increased in neurons and astrocytes, indicating that neuroprotection from IRI with this treatment probably occurs via astrocytes. Recent results indicate that astrocytes mediate pyruvate to attenuate glutamate neurotoxicity [38], and this protection occurs by altering expression of heat shock protein 72 and mitochondrial superoxide dismutase [39]. We demonstrated Nrf2 expression in the cytoplasm but not the nuclei of OKD mice, and this was because the OKD construct was designed to lack a DNA binding site [26]. In human cerebral ischemia, Nrf2 was activated in both neurons

Fig. 6. Scheme for BARD-mediated neuroprotection. Under normal conditions, Nrf2 is rapidly degraded by the ubiquitin–proteasome pathway through its association with Keap1. BARD dissociates Keap1 from Nrf2 by interacting with the Cys-151 residue of Keap1. The stabilized Nrf2 then translocates to the nucleus and activates the transcription of a wide range of cytoprotective genes, including HO-1, which protects neurons directly and via astrocytes.

T. Takagi et al. / Free Radical Biology and Medicine 72 (2014) 124–133

and astrocytes, supporting the hypothesis that Nrf2 protects neurons in the penumbra both directly and via astrocytes. Regarding the cell-type-specific expression of Nrf2, a previous study described expression in neurons but not glial cells [24]. On the other hand, Dang et al. [25] reported Nrf2 expression in neurons, astrocytes, and microglial cells. Collectively, the evidence suggests that Nrf2 is expressed in both neurons and astrocytes, and activation in both cell types is needed to protect neuronal cells from IRI. In conclusion, we demonstrated the time course of Nrf2 activity, visualized Nrf2 expression in the penumbra, and investigated cell-type-specific Nrf2 expression using OKD mice. Endogenous upregulation of Nrf2 was not shown early in ischemia; rather, it peaked at 24 h as demonstrated by in vivo imaging and Western blot, and Nrf2 was expressed mainly in neurons and astrocytes in the penumbra. Our results indicate that early activation of Nrf2 after ischemia onset has the potential to protect neurons from IRI via astrocytes. Intravenous administration of an Nrf2 activator (BARD) increased Nrf2 and HO-1 expression earlier, which protected neurons from IRI. Collectively, our results indicate that Nrf2 activators may be neuroprotective in IRI.

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