Therapeutic agents and reagents
SF and BP were obtained from Shanghai Institute of Materia Medica, Chinese Academy of Sciences. GIBCO® Dulbecco’s modified Eagle medium/F12 (DMEM/F12) was product of Life Technologies (USA). Primary antibodies for CD90-PE, CD45-FITC, CD34-PerCP were from Santa Cruz (California, USA). 2,3,5-triphenyltetrazolium chloride (TTC), MTT and Simvastatin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies of VEGF, von Willebrand factor (vWF), brain derived neurotrophic factor (BDNF) and neuronal class III β-tubulin (Tuj1) were products of Abcam (Cambridge, UK). Avidin–biotin peroxidase complex (ABC) kit and 3,3′-diamino-benzidine (DAB) kit were from Zymed Laboratories Inc (California, USA). Primary antibodies of AKT, p-Akt, mTOR and p-mTOR were obtained from Cell Signaling Technology, Inc. (Danvers, USA). The other materials and reagents were from commercial sources.
Primary culture of BMSC and identification
BMSCs were obtained and cultured as our previous report [16]. Briefly, the cells were harvested aseptically from bone marrow of the tibias and femurs in 50–60 g male Sprague–Dawley (SD) rats and cultured with DMEM/F12 cell medium supplemented with 10 % fetal bovine serum and 1 % penicillin–streptomycin in a cell culture flask at 37 °C and 5 % CO2. New culture medium was replaced once every 48 h. The cells were digested and passaged with 0.25 % trypsin (HyClone) during cell logarithm growth period with the cell fusion of 80 %.
To evaluate the BMSC purity, flow cytometry (Becton, Dickinson and Company, USA) was performed to identify CD90, CD45 and CD34 surface markers of cultured cells. The cells from passage 3 were collected and fixed with 4 % paraformaldehyde for 5 min and washed with 0.1 M phosphate-buffered saline (PBS). After that, they were adjusted to the density of 1 × 107/ml and were respectively incubated with fluorescence-conjugated antibodies including CD90-PE, CD45-FITC, CD34-PerCP and PBS (negative control) in a black chamber at 4 °C for 30 min. After washing with PBS, the cells were analyzed by a flow cytometer equipped with the Cellquest system (Becton, Dickinson and Company, USA).
The establishment of permanent middle cerebral artery occlusion model and experimental group definition
The study conforms to the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised in 1996 for scientific purposes. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Macau University of Science and Technology. Adult male SD rats (Guangdong Medical Laboratory Animal Center, Foshan, Guangdong, China) weighing 240–260 g were used in all experiments. Permanent middle cerebral artery occlusion (pMCAo) was established according to our previous method [16]. In brief, the rats were anaesthetized with 10 % (w/v) chloral hydrate (3.0 ml/kg, i.p.) and their body temperature was maintained at 40 °C by animal heating pad. 4–0 surgical nylon suture (length of 20–22 mm determined by body weight) coated with polylysine was inserted into the lumen of the right common carotid and advanced into the internal carotid artery until it obstructed the origin of the middle cerebral artery. Finally, the neurological function was evaluated using a 5-point scale neurological deficit score as reported by Longa and colleagues [19]. Only animals with the score of 2–3 were selected for the subsequent studies (0 = no deficit, 1 = failure to extend left paw, 2 = circling to the left, 3 = falling to the left, 4 = unable to walk spontaneously and consciousness). Sham operated animals underwent the same surgery but without nylon suture inserted.
One hundred and twenty MCAo rats were randomly divided into 5 groups (24 rats per group), including MCAo group, BMSC group, Simvastatin (Sim) + BMSC group (as positive control), SF + BP group and SF + BP + BMSC group. One millilitre of PBS or BMSC suspension solution (2 × 106 cells/ml) was intravenously injected into MCAo, SF + BP or BMSC alone, Sim + BMSC and SF + BP + BMSC groups via caudal vein at 3 h after operation; and 1 ml of PBS or SF (60 mg/kg) was intraperitoneally injected into MCAo, BMSC, Sim + BMSC group or SF + BP and SF + BP + BMSC groups once a day for continuous 7 days, the dosage SF were determined according to our previous report [17]; Simvastatin (1 mg/kg) [10] was gavaged daily for 7 days in Sim + BMSC group and BP (10 mg/kg) was subcutaneously injected into SF + BP and SF + BP + BMSC groups once a day for continuous 3 days respectively, the ratio of SF and BP dosage was defined according to preliminary experimental result (see Additional file 1).
TTC staining
To observe the volume of infarction zone in rat brain on the 7th day after treatment, TTC staining was applied. Rats (n = 5) in each group were deeply anaesthetized with 10 % (w/v) chloral hydrate, and the brains were removed quickly and placed at −20 °C for 15 min, sections of 2.0 mm thickness were cut and stained with 2 % solution of TTC at 37 °C for 30 min. The cross-sectional area of each slice in infarcted brain was measured using image analysis software (Image-Pro Plus Version 6.0, USA). The infarction volume was calculated by the corrected infarct volume (CIV). The formula: CIV (%) = [contralateral hemisphere volume−(ipsilateral hemisphere volume−infarct volume)]/contralateral hemisphere volume × 100 [20].
Immunofluorescence staining
On the 7th day after ischemic stroke, rats (n = 6) in each group were anaesthetized with 10 % chloral hydrate. Brains were fixed by transcardial perfusion with saline, followed by perfusion and immersion in 4 % paraformaldehyde. Fresh frozen sections of 5 μm thickness were cut on a cryostat microtome (Thermo fisher Scientific Shandon Cryotome FSE). Every 5th coronal section for a total of 6 sections was used for immunofluorescence staining. Prior to all staining procedures, tissue slices were extensively rinsed with 0.02 M PBS and blocked with 5 % normal goat serum in 0.02 M PBS containing 0.3 % Triton X-100 for 1 h. To identify the expressions of VEGF and BDNF in the IBZ, the slices incubated with rabbit primary antibodies to VEGF (1:50) and BDNF (1:200) at 4 °C overnight, followed by secondary antibodies to Alexa Fluor 488 labeled goat anti-rabbit IgG (H + L) (Life Technologies, USA) at 37 °C for 1 h. Nuclei were finally stained with 4′,6-diamidino-2-phenylindole in all images. The slices were visualized and digitally photographed with a confocal laser scanning microscope (Zeiss LSM710, Germany), 5 non-overlapping fields of one slice were randomly observed under a magnification of 10 × 40 in confocal images. To quantitatively analyze the proteins expression, Image-Pro Plus software was applied.
Histological and immunohistochemical staining
Rat brains (n = 6) were fixed with 4 % paraformaldehyde, and embedded in paraffin and cut into a series of 6 μm thick sections. For a morphological analysis of vessels and newborn neurons, samples were rinsed with Dulbecco’s phosphate buffered saline (Sigma, USA) containing 0.01 % Tween-20 and immersed in 3 % H2O2/methanol for 15 min to inhibit endogenous peroxidase activity. Subsequently, brain sections were incubated with 10 % normal goat serum for 20 min at room temperature, and then incubated with primary antibodies against vWF (1:100) and Tuj1 (1:200) at 4 °C for overnight. Following incubation with secondary antibody and ABC kit, sections were colored with DAB kit, and then stained with hematoxylin as a counterstain. Five slides were taken from each brain and each slide was randomly chose 5 non-overlapping fields to observe the expressions of vWF and Tuj1 under BX51 microscopy (Olympus, Japan). The determinant of vascular density was assessed by the number of positive vWF-vessels/mm2 using the Aperio ImageScope 12.3 (Leica), simultaneously vWF positive vascular perimeter and Tuj1 expression were quantified using Image-Pro Plus software in IBZ.
MTT assay and experimental group divided in vitro
In order to find the optimal dosage of SF and BP treatment for BMSC, the cells (1 × 105 cells/ml) were seeded in 96-well plates and incubated with different concentrations of SF (400, 200, 100, 50, 25, 5, 1, 0.1 and 0.01 μg/ml), BP (4, 2, 1, 0.75, 0.5, 0.25, 0.125, 0.01 and 0.001 μg/ml) for 48 h. According to the results, we found the SF concentration of 1 μg/ml and the BP concentration of 0.75 μg/ml had significant capacities of enhancing the viability of BMSCs, subsequently, we set the different concentrations of SF (50, 40, 20, 15, 10, 5 and 1 μg/ml) combined with BP (0.75 μg/ml) to incubate with BMSCs for 48 h. Prior to each assay, culture medium in each well was replaced by 100 μl fresh medium containing 10 μl of 5 mg/ml MTT stock solution. After 4 h of labeling cells with MTT, medium was removed and replaced with 100 μl DMSO in each well for 10 min at 37 °C. Samples were mixed and absorbance was set at 540 nm by using an enzyme-linked immunosorbent assay reader [18]. Twelve replicated wells were included in each group, and means were calculated. Finally, when we decided the optimal dosage of SF combined with BP treatment for BMSC, four groups were established which included normal group as control, SF (5 μg/ml) group, BP (0.75 μg/ml) group and SF + BP group.
Western blot analysis
Western Blot assay was conducted according to our previously described method [17]. The protein samples came from four groups’ BMSCs incubated with different therapeutic agents after 24 h in vitro and cortex of ischemic hemisphere of each group (n = 7) on the 7th day after the operation in vivo, and protein concentration was determined by enhanced BCA protein assay Kit (Beyotime Institute of Biotechnology, Shanghai, China). The samples were electrophoresed on sodium dodecyl sulphate–polyacrylamide gel and electrophoretically transferred to PVDF membranes in Tris–glycine transfer buffer. Then, membranes were blocked with 5 % (w/v) instant non-fat dried milk for 1 h at room temperature, and incubated with rabbit primary antibodies corresponding to β-actin (internal control) (1:1000), VEGF (1:1000), BDNF (1:100), AKT (1:1000), p-AKT (1:2000), mTOR (1:1000) and p-mTOR (1:1000) at 4 °C overnight. The membranes were subsequently washed with TBST [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.05 % Tween 20] and then incubated with secondary goat anti-rabbit IgG (H + L) (1:5000; LI-COR, USA) for 1 h at room temperature. The bands were visualized with Odyssey Infrared Imaging System (LI-COR, USA). The expressions of VEGF and BDNF were normalized against that of β-actin, phosphorylated levels of AKT and mTOR were analyzed by total levels of corresponding protein. The assay was replicated for three independent times.
Statistical analysis
Statistical calculations were performed with Statistical Product and Service Solutions (SPSS) (version 17.0, Chicago, IL, USA) by one-way analysis of variance followed by least significant difference test for multiple comparisons. Data were expressed as mean ± standard deviation (SD). Differences were considered to be statistically significant at p < 0.05.
