Lysates was measured using a caspase-3 colorimetric protease assay kit (Thermo
Lysates was measured using a caspase-3 colorimetric protease assay kit (Thermo Scientific). The results were expressed as the relative percentage of caspase-3 activity of each sample compared with the control cells.AnimalsMale Sprague awley rats, weighing 260?80 g, were housed in cages (45 ? 30 ? 20 cm3) on 12-h light/dark cycle with ad libitum access to chow and water. The animal studies were performed following the ARRIVE guideline (Additional file 2). All of the experimental animal procedures used in this study were conducted with the approval of the Ethics Committee of the Chinese University of Hong Kong (AEEC ref number: 11/068/MIS-5) (Additional files 3, 4, 5).Middle cerebral artery occlusion (MCAO)The SOD activity in the PC12 cells after OGD was measured using a SOD assay kit (Cayman Chemical, Ann Arbor, MI, USA). Tetrazolium salt, xanthine oxidase and cell lysate were added to a 96-well plate. The OD540 value of the formazan produced was measured by a fluorescent microplate reader (FLUOstar OPTIMA, BMG LABTECH, Ortenberg, Germany). The results of these experiments were expressed as the relative percentage of SOD activity of each sample compared with the control.Glutathione peroxidase assayThe GPx levels in the PC12 cells after OGD were measured using a Glutathione Peroxidase assay kit (Cayman Chemical) according to manufacturer’s instructions. The activity of GPx was estimated by measuring the optical density at 340 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28045099 nm (OD340) using a Quant system (BioTek).Catalase assayThe catalase activity of the PC12 cells was measured after OGD and the subsequent drug treatment processes using a catalase fluorometric detection kit (Enzo Life Sciences, Farmingdale, NY, USA), following the manufacturer’s instructions. The optical density at 595 nm (OD595) of resorufin was recorded. The results were expressed asRats were arbitrarily divided into three groups: GUW treatment, Control and Sham groups. After MCAO or a sham operation, the rats in the control and sham groups were orally administered with distilled water, whereas the rats in the GUW treatment group were administered with 288.6 mg/kg GUW once daily by oral gavage using an intragastric tube for 7 consecutive days. Sprague awley rats were intraperitoneally anesthetized with 400 mg/kg of chloral SIS3 supplier hydrate (VWR International Ltd, Poole, England). The cerebral blood flow (CBF) in these animals was intraoperatively monitored by laser Doppler flowmetery (PeriFlux System 5000, Perimed AB, Stockholm, Sweden). The probe holder (Probe holder PH07-6, Perimed AB) was located at the right cerebrum at 1 mm posterior and 2 mm lateral to the bregma above the skull. Briefly, the external carotid artery (ECA) and its branches were isolated and ligated. A round tip 4-0 nylon suture was inserted into the internal carotid artery (ICA) through the opening at the ECA and advanced to the anterior cerebral artery to occlude the middle cerebral artery (MCA). Once the MCA was occluded, the CBF was reduced by 60 . After 2 h of occlusion-induced ischemia, reperfusion was allowed by the gentle removal of the nylon filament. For the sham rats, the left CCA and ECA were exposed without inserting the filament into the ICA.Xian et al. Chin Med (2016) 11:Page 5 ofInfarct volume assessmentBrain infarct volume was quantified by 2,3,5-triphenyltetrazolium chloride (TTC) staining [30, 31]. Rats were euthanized with an overdose of pentobarbital at 7 days post-operation. The brains of the rats were harvested and cut i.