The investigation conformed to the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication, No. 86-23, revised 1985). The investigators responsible for molecular, histological and functional studies were blinded to the treatment groups.
Preparation and characterization of MSCs
MSCs were prepared from rat bone marrow as described by Friedenstein et al . In brief, we euthanized Sprague Dawley (SD) rats weighted 80-100 g and harvested bone marrow. Bone marrow cells were introduced into 100-mm dishes and cultured in complete medium, consisting of Dulbecco's Modified Eagle's Medium (DMEM; Sigma) containing 10% fetal bovine serum and antibiotics: 100 U/ml penicillin G, 100 mg/mg streptomycin, and 0.25 mg amphotericin B. Culture medium was replaced every three days and floating cells were discarded. Following two passes, the attached cells were divided into three new flasks and cultured until the cell density of the colonies grew to approximately 90% confluence. These cells were analyzed by fluorescence-activated cell sorting (FACS) as described previously . After blocking for nonspecific binding with buffer containing 1% bovine serum albumin, the cells were incubated for 20 minutes at 4°C with the following antibodies: anti-CD29, Phycoerythrin (PE), anti-CD106, PE, (Biolegend). anti-CD44, luorescein isothiocyanate (FITC), anti-CD14, FITC and anti-CD45, FITC (AbD Serotec). The matched isotype controls were purchased from AbD Serotec or Biolegend. At least 1 × 104 cells per sample were acquired and analyzed.
MSCs differentiation assay
The differentiation of MSCs in vitro towards the adipogenic and the osteogenic lineage as previously described [11, 12]. Briefly, for adipocyte differentiation, MSCs was cultured 3 weeks with adipogenic medium, containing 10-6M dexamethasone, 10 μg/ml insulin and 100 μg/ml 3-isobutyl-1-methylxantine (Sigma). For Osteoblast differentiation, MSCs was cultured 3 weeks with osteogenic medium, containing 10-7M dexamethasone, 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate (Sigma). Oil-red-O and von kossa dyes were employed to identify adipocytes, osteoblasts respectively.
SVV recombinant lentiviral vector construction
Human SVV recombinant lentiviral vector was constructed using previous method . Briefly, the full-length human SVV cDNA without termination codon was amplified by polymerase chain reaction (PCR) from pUC18-SVV and inserted into the Age I site of the GCFU plasmid to form a GFP/SVV fusion gene. The identity of SVV cDNA obtained in this manner was confirmed by sequencing and comparing it with the Gene Bank sequence NM_001168.2. The primer sequence was forward, 5'-GATGATGACGACAAACCGGTCATGGGTGCCCCGACGTTG-3' and reverse, 5'-TCACCATGGTGGCGACCGGTTTATCCATGGCAGCCAGCTG-3'. The SVV recombinant lentiviral vector was prepared using Lipofectmaine 2000 transfection technology.
MSCs gene modification
For passage 1 MSCs were infected by lentivirus with a multiplicity of infection (MOI) of 8 . The MSCs infected with SVV recombinant lentivirus were defined as SVV/GFP-MSCs and the MSCs infected with mock lentivirus were defined as GFP-MSCs. To achieve the optimal gene transfer, polybrene (a final concentration of 8 μg/ml) was used. All MSCs were expanded to 3 passes, and then used for transplantation. The efficiency of gene transduction was assessed with FACS.
SVV expression in modified MSCs
The survivin expression was detected by immunofluorescence staining. In brief, the 3rd passage transfected MSCs were plated onto fibronectin-coated chamber slides, fixed with 4% paraformaldehyde (Sigma) for 10 minutes at room temperature, and washed twice in 0.01 M phosphate-buffered saline (PBS, GIBCO). Slides were blocked with goat serum for 20 minutes and incubated overnight with mouse anti-human Survivin antibody (AbCam) at 4°C. After that, the slides were incubated with Texas-Red fluorescent anti-mouse secondary antibody (Santa Cruz) for 30 minutes at 4°C. Between steps the slides were washed with PBS. A 1:500 dilution of primary antibody against human SVV and a 1:500 dilution of secondary antibody were used, respectively. Cells were examined by fluorescencemicroscopy (Leica Co, Germany).
Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) secretion in MSCs under hypoxic conditions
After the 3rd passage infected MSCs completed adherence, they were incubated for 24 hours at 37°C in a humidified modular hypoxia chamber (Billups Rothenberg) containing 95% nitrogen and 5% carbon dioxide (n = 4 in each group). Subsequently, the supernatants were collected for analysis. Commercial VEGF or bFGF ELISA (enzyme-linked immunosorbent assay) kits (R&D Systems Inc. Minneapolis, USA) was used to quantify the concentration of VEGF and bFGF in each of the samples. The supernatant from MSCs cultured in normal condition was used for control. Any experiment was repeated for three times.
Adult male Sprague-Dawley rats weighing 220-250 g were used in this study. A middle cerebral artery occlusion (MCAO) was established with the modified Longa method . Rats were initially anesthetized with 10% chloral hydrate. Rectal temperature was controlled at 37°C with a feedback-regulated water heating system. The right common carotid artery, external carotid artery (ECA), and internal carotid artery were exposed. A 3.0 monofilament nylon suture (18.5 mm, determined by animal weight), with its tip rounded by heating near a flame, was advanced from the ECA into the lumen of the internal carotid artery until it blocked the origin of the middle cerebral artery (MCA). 2 hours after MCAO, animals were reanesthetized with halothane, and reperfusion was performed by withdrawal the suture until the tip cleared the lumen of the ECA.
MSCs transplantation was performed as a method reported in previous study . Briefly, after 2-hour middle cerebral artery occlusion (MCAO) and 24-hour reperfusion, Rats were grouped into three groups which received a 500 μl injection of either phosphate-buffered saline (PBS) without cells (group control, n = 18) or containing three million GFP-MSCs (group GFP, n = 30) or SVV/GFP-MSCs (group SVV, n = 30) via tail vein.
Double Immunofluorescence Staining
In order to identify survival and differentiate of the transplanted MSCs, a method of double immunofluorescent staining was used. Rats in the GFP and SVV groups were euthanized with 10% chloral hydrate at 4 days (n = 6 in each group) or 14 days (n = 6 in each group) after transplantation. For preparation of frozen sections, rats were perfused transcardially with normal saline and the brain samples were removed immediately. Blocks corresponding to coronal coordinates form bregma -1 to 1 mm were obtained and frozen rapidly in liquid nitrogen. A series of 6-um-thick sections was obtained. Thereafter, the frozen sections were rewarmed at room temperature for 45 minutes to 1 hour, and were concubated overnight at a dilution of 1:200 with FITC labeled goat anti-GFP (AbCam) and rabbit anti-rats Neuronal nuclei (NeuN, which is a marker of neuron.) (DAKO), and then incubated for 45 minutes using a secondary antibody of goat anti-rabbit/mouse IgG conjugated with TAXES (Santa Cruz) for detecting NeuN at 37°C. Between steps the slides were washed with 0.01M PBS. Finally, the sections were used to detect the survival and differentiation into neuron-like cells of the transplanted MSCs by a laser scanning confocal microscope (Zeiss Co., LSM510).
Western Blot for VEGF and bFGF in Injuried Cerebral Tissues
Rats were euthanized with 10% chloral hydrate at 4 days (n = 6 in each group) or 14 days (n = 6 in each group) after transplantation. The protein concentration from injured cerebral tissues was determined using the bicinchoninic acid (BCA) protein assay kits (Beyotime Biotechnology, P.R. China). Thirty micrograms protein were loaded on 10% acrylamide gel for electrophoresis and were electroblotted onto a polyvinylidene difluoride membrane (PVDF, Invitrogen). The membranes were then probed with mouse anti-VEGF (1:500) and anti-bFGF (1:500), respectively, followed by incubation with horseradish-peroxidase-conjugated sheep-anti-mouse IgG (Bio-Rad Laboratories). Protein expression was detected with an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech Inc) and β-actin was used as a loading control. All bands from western blot were analyzed using Image J software (version 1.6 NIH) to verify the relative level of VEGF and bFGF defined as the optical density ration of VEGF or bFGF over β-actin.
Measurement of Cerebral Infarction Volume
At 14 days after MSCs transplantation, rats in each groups (n = 6) were used for evaluate cerebral infarction volume. The brain samples were removed carefully and dissected into five equally spaced coronal blocks using a vibratome. The fresh brain slices were immersed in a 2% solution of 2, 3, 5-triphenyltetrazolium chloride (TTC) (Sigma) in PBS (GIBCO) at 37°C for 30 minutes. The cross-sectional area of infarction and non infarction in each brain slice was measured using Image J analysis software (version 1.6 NIH). The infarct volume was indirectly determined by subtracting the volume of intact tissue in the ipsilateral hemisphere from that in the contralateral hemisphere.
Evaluation of neurological function
Evaluation of neurological function was performed 1 day and 14 days after transplantation in each groups (n = 6) using a modified Neurological Severity Score (mNSS) . The mNSS is a composite of the motor (muscle status and abnormal movement), sensory (visual, tactile, and proprioceptive), and reflex tests. The neurological function was graded on a scale of 0-18 (normal score 0, maximal deficit score 18)
Data were presented as mean values and standard deviation. A method of ANOVA (analysis of variance) with Scheffe's post hoc test was used to identify differences among all groups. A P value of less than 0.05 was considered as statistical significance.