All experimental procedures were approved by Institutional Animal Care and Use Committee of Anhui Medical University. Mice (male, 10–12 weeks) were kept on a 12/12 hours day and night cycle and with food and water available Ad libitum. Mice were anesthetized with isoflurane using anaesthesia machine (Matrx, Orchard Park, NY, USA). Transient focal cerebral ischemia was induced by intraluminal occlusion of the right middle cerebral artery via silicone-coated nylon monofilament (6023PK; Doccol Corp, Redlands, CA, USA). Briefly, for the transient model, reperfusion was produced by withdrawal of the monofilament 1 hour after occlusion in mice. The regional cerebral blood flow was continuously assessed by recording laser Doppler flow (Moor VSM-LDF; Moor Instruments, Wilmington, DE, USA) to ensure appropriate ischemia and reperfusion. Body temperature was regularly checked and maintained between 36.5°C and 37.5°C with a heating pad to avoid hypothermia. The sham-operated animals underwent the same anesthesia and surgical procedures except tMCAO.

GB (from National Institutes for Food and Drug Control, China) was freshly diluted to the concentrations as needed with 0.9% saline and administered intraperitoneally 60 minutes after tMCAO. All mice were randomly assigned to the following experimental treatment groups: a sham-operated group (saline, 10 mL/kg), a GB-only group (GB at doses of 4 mg/kg, i. p., bid.), a tMCAO group (mice suffered from tM-CAO and saline, 10 mL/kg), and tMCAO+GB groups (mice suffered from tMCAO, followed by GB at doses of 1, 2, and 4 mg/kg, i. p., bid.). The doses of GB were comparable with therapeutic concentration in humans.

Neurological deficits were assessed 72 hours after tMCAO described using a 5-point-scale neurological score2): 0, normal motor function; 1, flexion of the contralateral torso and forearm on lifting the animal by the tail; 2, spontaneous circling to the contralateral side but normal posture at rest; 3, leaning/falling to the contralateral side; 4, no spontaneous movement; 5, death.

At 72 hours after tMCAO, the mice were deeply anaesthetized with pentobarbital sodium (80 mg/kg) and euthanized by decapitation. The brains were quickly removed and sectioned coronally into slices of 1 mm thickness. Slices were incubated in a 1% solution of 2, 3, 5-triphenyltetrazolium chloride (TTC; Sigma, St Louis, MO, USA) in phosphate buffered saline at 37°C (water bath) for 15 minutes and then fixed by 4% buffered formaldehyde solution. TTC stained viable brain tissue dark red based on intact mitochondrial function, whereas infarcted tissue areas remain unstained (white). Images were acquired by a digital camera (Nikon Coolpix; Nikon Corporation, Tokyo, Japan), and areas of both hemispheres and the infarcted regions were quantified for each slice using Image J software (National Institutes of Health, Bethesda, MD, USA). The actual infarct volume with correction of edema was calculated as the volume of the contralateral hemisphere minus the noninfarcted volume of the ipsilateral hemisphere. Edema was measured using the wet/dry weight method with water content expressed as the percent of tissue wet weight as previously described8).

N2a cell, an immortalized cell line of murine nerve cells, was purchased from American Type Culture Collection. Cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (Millipore, Billerica, MA, USA), and 1% penicillin and streptomycin in a 37°C humidified incubator with 21% O₂ and 5% CO₂. To generate OGD/R model, cells were incubated with glucose-free medium (Gibco) in a 37°C incubator chamber (Thermo Scientific Forma^® Series II Water Jacketed CO₂ Incubators model 3131; Thermo Fisher, Waltham, MA, USA) aerated with an anaerobic gas mixture (1% O₂, 5% CO₂, 94% N₂). After 3 hours, cells were moved out from the anaerobic chamber and then the glucose-free medium was replaced with normal medium. The cells were cultured for 24 hours in normal incubator for re-oxygenation. GB (0.1–40 μM) was pre-incubated 2 hours before OGD/R injury.

Cell viability was determined by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide incorporation. The optical density value was measured at 570 nm and 630 nm using micro-plate reader (Bio-Rad, Hercules, CA, USA). Cell injury was assessed by measuring the activity of lactate dehydrogenase (LDH) in the culture medium. Data were presented as percentage of the maximal LDH release.

For analysis of specific proteins abundance 72 hours after stroke or 24 hours after OGD/R, ischemic penumbra of individual mice or cell homogenate was complemented with lysis buffer according to instructions of the protein extraction kit (Nanjing KeyGen Biotech. Co. Ltd., Nanjing, China). The supernatants were used for sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes with the electrophoretic transfer system (Transblot Semi-dry Transfer Cell; Bio-Rad). The membranes were blocked with blocking solution (10% milk in 0.1% Tween 20 Tris-buffered saline; 1 hour at room temperature) and incubated with primary antibodies including BDNF (AB6201; Abcam, Cambridge, MA, USA), Bcl-2, Bax, Caspase-3 (1: 1000; Cell Signaling Technology, Danvers, MA, USA), and β-actin (1: 2000; Sigma) overnight at 4°C. Thereafter, membranes were incubated with corresponding secondary antibodies (1: 1000; Invitrogen, Waltham, MA, USA) at room temperature. Finally, immunoblots were scanned and determined by Omega16IC (Ultra-Lum, Inc., Claremont, CA, USA).

Results were presented as mean±standard error of mean and analyzed with one-way analysis of variance (ANOVA). Neurological scores (median value) were analyzed by Wilcox-on-Mann-Whitney test. For all analyses, p<0.05 was considered statistically significant.

To examine the neuroprotective effect of GB in vivo, GB (1, 2 or 4 mg/kg, i. p., bid) was administered 1 hour after onset of reperfusion in tMCAO mice. Neurological deficits and brain water contents were measured 72 hours after tMCAO. As shown in Fig. 1, GB could significantly reduce ischemia-induced neurological deficits (Fig. 1A) and brain water content (Fig. 1B) at dose of 4 mg/kg.

As shown in Fig. 2, GB significantly decreased total infarct volume at dose of 1, 2, and 4 mg/kg (by 21.2%, 34.5%, and 52.6% respectively, p<0.05) in comparison with mice received saline treatment 72 hours after reperfusion. These results demonstrated that GB significantly alleviated cerebral ischemia/reperfusion-induced brain damage in vivo.

To assess whether GB can regulate BDNF expression, we evaluated the protein expression of BDNF in the ischemic penumbra of individual mice. As shown in Fig. 3, we observed that GB (4 mg/kg) significantly increased the expression of BDNF protein (p<0.05).

To further investigate whether GB produce a cytoprotective effect against ischemic insult, we generated an OGD/R model using N2a cells. Pre-treatment with GB significantly promoted the viability of cells (Fig. 4A), and decreased the release of LDH (Fig. 4B) in a concentration-dependent manner after OGD/R.

To determine the effects of GB on OGD/R-induced cell apoptosis, the proteins Bcl-2, Bax, and Caspase-3 were calculated in the presence or absence of OGD/R injury. As shown in Fig. 5, GB significantly increased Bcl-2/Bax ratio, and reduced the expression of caspase-3. Furthermore, in accordance with the results in vivo, OGD/R stimulation decreased the expression of BDNF, while the administration of GB induced a conspicuous increase. These observations indicated that GB may effectively alleviate OGD/R-induced apoptosis via upregulating BDNF expression.