All animal experiments were approved by the Institutional Animal Care and Use Committee of National Cheng Kung University (Tainan, Taiwan). Cultured neurons were obtained from cerebral cortices of 2 day-old Sprague-Dawley rats (n=48; purchased from the Research Animal Care of National Cheng Kung University Medical Center) according to methods described previously (19,20). Rats were housed at 24±1°C, 60% humidity and on a 12 h light/dark cycle. The neonatal rats received nutrition from their mother. Following sacrifice, the cortices were separated in ice-cold Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Tissue chunks were crushed and incubated with Hank's Balanced Salt Solution (HBSS; Gibco; Thermo Fisher Scientific, Inc.) containing papain (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and DNase I (Bionovas Biotechnology Co., Ltd., Toronto, OΝ, Canada) at 37°C to dissociate the cells. After 30 min incubation, 10% heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) was added to cell suspensions to terminate the reaction. Cells were then were centrifuged at 4°C, 800 × g for 10 min. The pellets were collected and suspended in DMEM with 10 % FBS, and then were filtrated using a 70 µm cell strainer (BD Biosciences, Franklin Lakes, NJ, USA). Dissociated cells were plated onto poly-D-lysine-coated petri dishes and incubated at 37°C in a humidified incubator with 5% CO₂. A total of 4 h after plating, the culture medium was replaced by neurobasal medium containing 25 µM glutamate, 0.5 mM L-glutamine, and 2% B27 supplement (Invitrogen; Thermo Fisher Scientific, Inc.). The culture medium was replaced every 3 days. Cultured neurons were allowed to grow for 7–14 days.

The model of stroke in primary cortical neuronal culture cells were achieved by combining hypoxia with aglycemia according to a method described previously (19). The OGD medium consisted of HBSS that was lacking glucose and that had previously been infused with N₂ for 30 min. The cultured neurons were pretreated with dimethyl sulfoxide (0.1% DMSO; Sigma-Aldrich; Merck KGaA) as a vehicle, or 30 mM YC-1 for 30 min, and then were incubated with OGD medium and transferred to an anaerobic chamber at 37°C in an N₂-enriched atmosphere for 2 h. Following the deprivation period, cultured neurons were incubated in neurobasal medium under normal conditions (a humidified incubator with 5% CO₂ at 37°C).

EMSA was performed according to a method described previously (21). Nuclear proteins were extracted using an NE-PER Nuclear Protein Extraction kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. An EMSA assay was carried out using a digoxigenin (DIG) gel shift kit (Roche Applied Science, Penzberg, Germany). DIG was labeled with 3.85 pmol/µl NF-κB Gel Shift Oligonucleotides with the following primer sequences: Forward, 5′-AGTTGAGGCGACTTTCCCAGGC-3′ and reverse, 3′-TCAACTCCGCTGAAAGGGTCCG-5′ (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Nuclear protein (10 µg) was incubated with a DIG-labeled probe for 30 min on ice, electrophoresed on a 6% polyacrylamide gel and subsequently transferred onto a hybridization transfer membrane (PerkinElmer, Inc., Waltham, MA, USA). The DIG-labeled probe was recognized by an alkaline phosphatase-conjugated anti-DIG antibody (1:5,000; cat. no. 03353591910; DIG gel shift kit, Roche Applied Science) at room temperature for 30 min and was detected by a luminescent image analyzer (Fujifilm LAS-3000; Fuji Photo Film Co., Tokyo, Japan).

Cultured neurons (at days 7–14) were pretreated with vehicle (DMSO) or YC-1 (30 µM) for 30 min, and then were exposed to OGD for 2 h. The supernatants were collected 48 h post-treatment and were stocked at −20°C until required. Samples (10 µl) were mixed with sample buffer and loaded onto a 7.5% SDS-PAGE gel containing 0.125% gelatin (Sigma-Aldrich; Merck KGaA). Following electrophoresis, the gel was washed with 2.5% Triton X-100 buffer (J.T. Baker, PA, USA) three times. The gel was incubated in 100 ml 50 mM Tris-HCl (pH 7.4) containing 5 mM CaCl₂, 200 mM NaCl, and 0.2% Brij 35 (Sigma-Aldrich; Merck KGaA) at 37°C overnight. The gel was stained with 0.25% Coomassie blue R-250 (Sigma-Aldrich; Merck KGaA) at room temperature for 60 min.

Nuclear and cytoplasmic proteins were extracted by using the NE-PER Nuclear Protein Extraction kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The protein concentration of each sample was determined with a Bicinchoninic Acid protein assay kit (Thermo Fisher Scientific, Inc.). Sample proteins (20–50 µg) were separated by 10% SDS-PAGE and subsequently transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% skimmed milk in phosphate buffered saline with 0.5% Tween-20 (PBST) buffer for 30 min at room temperature, and then were incubated with primary antibodies against phosphorylated (p)-IκB-α (cat. no. sc-8404; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and NF-κB (ADI-KAS-TF110-D; 1:500; Enzo Life Sciences, Inc., Farmingdale, NY, USA) at 4°C overnight. After washing, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (cat. no. AP192P; anti-mouse; cat. no. AP307P; anti-rabbit; 1:2,000; Merck KGaA) for 1 h at room temperature, and then washed with PBST. Proteins were visualized with an Enhanced Chemiluminescence kit (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Membranes were then probed for GAPDH (cat. no. GTX100118; 1:500; GeneTex, Inc., Irvine, CA, USA) or transcription factor II D (cat. no. sc-204; 1:200; Santa Cruz Biotechnology, Inc.) as an internal control. Optical densities were measured using Multi Gauge V3.0 (Fuji Photo Film Co.) on a Luminescent Image Analyzer (Fujifilm LAS-3000; Fuji Photo Film Co.).

Adult male C57 black (C57BL/B6) mice, (age, 8 weeks; n=34; weight, 20–26 g; obtained from Research Animal Care of National Cheng Kung University Medical Center), were housed at an ambient temperature of ~24±1°C and were allowed free access to food and water prior to and following surgery. For the surgical procedures on mice, 1–2% halothane (Sigma-Aldrich; Merck KGaA) in N₂O:O₂ (70:30%) was used for anesthesia. The study was approved by the Research Animal Care of National Cheng Kung University Medical Center.

Focal cerebral ischemia was employed by intra-arterial suture occlusion of the proximal right middle cerebral artery (MCA) according to a method described previously (22). Briefly, the external carotid artery (ECA), internal carotid artery (ICA) and the pterygopalatine artery of the ICA were exposed under an operating microscope. A silicone rubber coated nylon suture was inserted into the ICA via a slit in the ECA. The suture was advanced 7.5–8.5 mm along the ICA until the tip occluded the origin of the MCA. Reperfusion was produced by gently removing the suture and closing the incision after 60 min of MCA occlusion (MCAO). Following surgery, the animals were kept in a cage with a heating lamp, monitored for 2 h and then transferred into the home cage.

YC-1 was dissolved in a mixture of PEG 400 (30% PEG 400 in saline). Animals were intravenously administered with either 5 or 25 mg/kg YC-1, or vehicle (PEG 400) 60 min before surgery. The zymographic and in situ zymographic were examined after 24 h following ischemia reperfusion and brain infarction. Immunofluorescence and immunohistochemistry were performed 48 h after ischemia reperfusion.

All assessment was based on a method described previously (22,23). Body weight was measured daily. A variety of sensory-motor tests were conducted 48 h after ischemia-reperfusion. Two neurological grading systems were used: A sensorimotor grading scale and a grading scale of 0–28. The score given to each mouse at the completion of testing was the summation of seven individual test scores (body symmetry, gait, climbing of 45 angle plate, circling behavior, front limb symmetry, compulsory circling and whisker response). Locomotor impairment was assessed using a rota-rod treadmill (model 47700; Ugo Basile Biological Research Apparatus, Varese, Italy). Latency to fall off the rota rod was determined 48 h after stroke. Mice were conditioned to a rota-rod initially with a fixed speed that was set at 5 rpm for 1 min. A total of 10 min after the completion of the fixation rota-rod test, the speed was accelerated from 2 to 10 rpm over a period of 5 min. Each test was repeated three times, and the time spent on the rod was recorded. Mice that remained on the rota-rod after 5 min were given a maximum score of 300 sec.

All assessment was based on a method described previously (22). After 24 (n=14) or 48 h (n=20), mice were sacrificed under anesthesia (4% chloral hydrate/1 ml/100 g; Sigma-Aldrich; Merck KGaA) and perfused transcardially with 4% formaldehyde in 0.1 M PBS following ischemia-reperfusion. After postfixation and dehydration, the brains were embedded in Optimal Cutting Temperature compound (Tissue-Tek; Miles Laboratories Inc., Elkhart, IN, USA) and frozen in liquid nitrogen. The brains were sectioned into 10- or 40 µm pieces at eight preselected coronal levels on a cryostat (HM-500O; Microm International GmbH, Walldorf, Germany). Sections were mounted on poly-l-lysine-coated (Sigma-Aldrich; Merck KGaA) slides and dried at 37°C overnight, and then were stored at −20°C.

Brain sections (40 µm) were stained with 0.5% cresyl violet for 4 h at room temperature. Under a light microscope, the areas of neuronal perikarya displaying typical morphologic features of ischemic damage were delineated using a computerized image analyzer (MCID Elite; Imaging Research Inc., St. Catharines, ON, Canada). Infarct volumes were measured and expressed as a percentage of the contralateral hemisphere volume (the contralateral hemisphere area minus the ipsilateral non-ischemic hemisphere area, and then divided by the contralateral hemisphere area). The ipsilateral brain edema was expressed as a percentage index relative to the volume of the left hemisphere (ischemic hemisphere area divide by contralateral hemisphere area minus infarct volume).

Immunofluorescence staining was performed based on a method described previously (24). A total of 48 h following ischemic insult, brain sections were post-fixed in 4% paraformaldehyde in PBS for 5 min at room temperature and incubated with alcohol:acetic acid at a ratio of 2:1 for 30 min. Following washing, the brain sections were incubated with polyclonal mouse anti-p-IκB (cat. no. sc-8404; 1:200), polyclonal rabbit anti-NF-κB (cat. no. sc-109; 1:200) and polyclonal goat anti-MMP-9 (cat. no. sc-6841; 1:200) primary antibodies, all purchased from Santa Cruz Biotechnology, Inc., at 4°C overnight. Following this, the brain sections were incubated with corresponding fluorescein isothiocyanate-conjugated IgG secondary antibodies [cat. no. AP124F (anti-mouse IgG), 1:2,000; Chemicon International lnc., MA, USA; 111-095-003 (anti-rabbit IgG), 305-095-003 (anti-goat IgG), 1:5,000; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA], for 1 h at room temperature. Sections were mounted with Dapi Fluoromount-G^® (SouthernBiotech, Birmingham, AL, USA) for 10 sec at room temperature. A Zeiss Axioskop 2 Mot microscope equipped with a digital CoolSnap-Pro cf camera (Media Cybernetics, Inc., Rockville, MD, USA) was used to image brain sections. The density of the p-IκB-, NF-κB- and MMP-9-positive cells were calculated relative to the total number of DAPI-stained cells.

Immunohistochemistry staining was performed based on a method described previously (21). A total of 48 h following ischemic insult, brain sections were post-fixed in 4% paraformaldehyde in PBS for 5 min at room temperature and subsequently incubated with alcohol:acetic acid at a ratio of 2:1 for 30 min. Following washing, the brain sections were incubated at 4°C overnight with primary antibodies against mouse anti-rat monoclonal ED-1 (cat. no. 120405, 1:200; Serotec, Raleigh, NC, USA), anti-goat myeloperoxidase (cat. no. sc-16129, 1:200; Santa Cruz Biotechnology, Inc.) and anti-rabbit iNOS (cat. no. AB5382, 1:200; EMD Millipore, Billerica, MA, USA), and were subsequently developed using a DAB Peroxidase (HRP) Substrate kit (Sigma-Aldrich; Merck KGaA). Sections were co-incubated with hematoxylin for 3 min at room temperature. The protein expression of brain sections were measure using a Zeiss Axioskop 2 Mot microscope equipped with a digital CoolSnap-Pro cf camera and a semi-automated image analysis system (MCID Elite; Imaging Research Inc.).

The integrity of the BBB was investigated with Evans blue dye extravasation according to previous reports with a few modifications (14). Animals were intravenously administered with either 25 mg/kg YC-1 (n=7), or vehicle (PEG 400, n=7) 60 min prior to surgery. Following surgery, 0.5 ml 4% Evans blue dye (Sigma-Aldrich; Merck KGaA) solution in saline was administered intravenously. A total of 24 h after injury, animals were anesthetized and then sacrificed by transcardial perfusion with ice-cold PBS. The brain was dissected out and homogenized in 50% trichloro acetic acid (Sigma-Aldrich; Merck KGaA) solution. After homogenization, samples were centrifuged at 4°C, 20,000 × g for 10 min, the supernatants were collected and diluted with ethanol (1:3). The fluorescence was quantified at an excitation wavelength of 620 nm and an emission wavelength of 680 nm by a Fluoroskan Ascent^™ FL Microplate Fluorometer (Thermo Fisher Scientific, Inc.). Dye in samples was determined as µg/g tissue from a standard curve plotted using known amounts of dye.

Data were analyzed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation. Unpaired Student's t-test or one-way analysis of variance followed by least significant difference post hoc comparison was used to evaluate differences between groups. Neurobehavioral scores were expressed as the median ±95% confidence interval and were analyzed by Mann-Whitney U test. *P<0.05 was considered to indicate a statistically significant difference.

In primary cortical neuronal cultures exposed to OGD, groups pretreated for 30 min with 30 µM YC-1, compared with controls, exhibited effectively reduced phosphorylation of the NF-κB inhibitory protein p-IκB in the cytoplasm. Additionally, YC-1 pretreatment significantly restored decreased NF-κB protein expression levels in cytoplasm, and attenuated the amount of NF-κB in the nucleus (Fig. 1A; P<0.05). The specific NF-κB binding activity was assessed by EMSA. YC-1 pretreatment significantly reduced upregulation of specific NF-κB binding activity (Fig. 1B; P<0.05). Additionally, C57BL/B6 mice were pretreated with a vehicle or 25 mg/kg YC-1 by intravenous injection 60 min before induction of transient focal cerebral ischemia. YC-1 treatment significantly reduced the density of p-IκB (Fig. 1C; P<0.05) and NF-κB (Fig. 1D; P<0.05) translocation in the area of brain injury at 48 h post-insult.

Adult male C57BL/B6 mice were subjected to transient focal cerebral ischemia. In the ischemic brain at 48 h post-insult, the 25 mg/kg YC-1-treated group exhibited a 52.5% reduction of iNOS-positive cells (Fig. 2A; P<0.05). Additionally, the YC-1 treated group exhibited significantly reduced numbers of neutrophils (Fig. 2B; P<0.05) and activated microglia/macrophages (Fig. 2C; P<0.05) by 37.1 and 36.5%, as assessed by the density of myeloperoxidase (MPO) and ED-1 positive cells, respectively.

In primary cortical neuronal cultures exposed to OGD, the vehicle-treated group demonstrated increased MMP-9 enzyme activation, compared with the control group. However, pretreatment with YC-1 significantly attenuated this activation, as assessed by gelatin-dependent zymography (Fig. 3A; P<0.05). However, no significant differences were observed in MMP-2 enzyme activation. Furthermore, in the ischemic brain at 48 h post-insult, YC-1 pretreatment significantly reduced the density of MMP-9-positive cells (Fig. 3B; P<0.05).

Evans blue dye was used as a marker to evaluate the effect of YC-1 on BBB permeability. Evans blue leakage increased significantly in the brains of control group mice subjected to MCAO and 24 h reperfusion, whereas administration of 25 mg/kg YC-1 to mice markedly ameliorated the Evans blue leakage (Fig. 4A; P<0.05).

Adult mice were subjected to transient focal cerebral ischemia. Compared with controls, 5 and 25 mg/kg YC-1 pretreated mice had infarction volumes reduced by 27.5 and 57.5%, respectively, and individual cortical lesion sizes reduced by 68.1% in the 25 mg/kg YC-1-treated group at 48 h post-insult. Furthermore, 25 mg/kg YC-1-treated mice demonstrated significantly reduced brain edema at 48 h after reperfusion (Fig. 4B; P<0.05).

Administration of 5 and 25 mg/kg YC-1 to mice by intravenous injection resulted in significantly improved sensory, motor and 28-point neurologic scores taken 48 h post-insult compared with the control group. Furthermore, the 25 mg/kg YC-1-treated group demonstrated a longer running time in the rota-rod motor performance test (Table I; P<0.05).