Animals
In the present study, male Sprague-Dawley (SD) rats (80-120 g) were used for MSC isolation and culture. SD rats (260-300 g) were used for middle cerebral artery occlusion (MCAO) model establishment. All rats were provided by Zhejiang Chinese Medical University Animal Center (Laboratory Animal Certificate: SYXK 2013-0184). Rats were raised in individual cages in a temperature- and humidity-controlled room with a 12-h light/dark cycle and access to food and water ad libitum.
MSC culture and pretreatment
MSCs were isolated from SD rats (80-120 g) under sterile environment as previously reported [16]. The long bone was taken from the hind legs of SD rats, and both ends of the long bones were cut. Bone marrow was flushed out with phosphate buffered saline (PBS, BOSTER, China). The obtained bone marrow solution was centrifuged at 1,000 rpm for 5 min, the supernatant liquid was removed, and the cell pellets were resuspended in Dulbecco’s Modified Eagle’s Medium/Ham's F-12 (DMEM/F12, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA). Cells were detached with 0.25% trypsin-ethylene diamine tetraacetic acid (EDTA, Gibco, USA) at 80% confluence. Passage 3 cells were used for the following experiments.
For hypoxia and ischemia, MSCs were pretreated with serum-free medium with 100 μmol/l of Cobalt chloride (CoCl2, Sigma, USA) for 12 h. For salidroside (HPLC ≥98%, Shanghai Yuanye Bio-Technology Co., Ltd., China) pretreatment, MSCs were cultured with different concentrations of salidroside (0.375, 0.75, 1.5, 3, 6 μg/ml) for 48 h, followed by exposure to 100 μmol/l of CoCl2 with serum-free medium for 12 h. The MSCs cultured in complete medium served as control. These pretreated cells were used for Cell Counting Kit (CCK)-8 assay, transwell assay, and flow cytometry assay.
CM-Dil (Invitrogen, Carlsbad, CA, USA) was used to label the cell membrane prior to transplantation, without affecting cell morphology, viability, and proliferation capacity. CM-Dil stock solution was prepared as recommended by the manufacturer.
CCK-8 assay
MSC proliferation was measured using the CCK-8 (Dojindo Laboratories, Inc., Kumamoto, Japan). MSCs were inoculated at 1 × 103 cells/well in 96-well culture plates. The medium was discarded, and each well was washed twice with PBS. One hundred microliters of DMEM/F12 and 10 µl of CCK-8 reagents were added to each well and incubated at 37°C with 5% CO2 for 2 h. The absorbance of the reaction solution was measured at 450 nm using a microplate reader (SpectraMax Plus384, Molecular Devices, LLC, Sunnyvale, CA, USA).
Transwell assay
Cell migration ability was performed using transwell plates (24-well plate, 8-μm pore size, Corning, NY, USA). Cells were digested and resuspended in DMEM/F12 containing 2% FBS. One hundred fifty microliters (1 × 105 cells/ml) of cell suspension was added to the upper chamber of the migration well, and 600 µl of complete medium was added to the lower chamber. After 15 h, cells from the top of the filter were carefully removed with a cotton swab. The cells that had migrated through to the underside of the insert membranes were then fixed with 4% paraformaldehyde for 30 min and stained with crystal violet for 10 min. Migrated cells were observed and imaged by a fluorescence microscope (Nikon, Eclipse Ts2-FL, Japan). The number of migrated cells was determined by averaging 6 random fields per well.
Flow cytometry assay
To quantify the apoptotic MSCs, cells were collected, washed with PBS, and then centrifuged at 1,000 rpm for 5 min before 1 × 105 cells were resuspended in the 1×AnnexinV binding buffer mixed with 5 μl of propidium iodide (PI) reagent. After incubation for 15 min at room temperature in the dark, another 400 μl of binding buffer was added, and cells were measured using a flow cytometry machine (Accuri C6, BD Biosciences)
MCAO model
MCAO was induced by the modified Zea Longa method [17]. Rats were anesthetized with 10% chloral hydrate (0.35 ml/100 g, Yonghua Chemical, China) and fixed on an operating table in a supine position. A midline neck incision was made, and the common carotid artery (CCA) and its bifurcation were exposed and isolated carefully from the vagus nerve. The bifurcation of the external common carotid artery and the proximal end of CCA were occluded by a silk suture. A small hole was cut into the CCA to insert a filament into the internal CCA. The suture was tightly tied around the filament to prevent bleeding and the reverse-action tweezers were removed. After 1.5 h of occlusion, the suture was withdrawn to enable reperfusion and the wound and skin were sutured. The rats in the sham group received the same procedures, except that the MCAO group was not occluded. Finally, animals were placed in a cage over a warming blanket until full recovery from anesthesia.
Transplantation of MSCs and animal grouping
Rats were randomly divided into 4 groups, including the sham, vehicle, MSCs (MSCs transplantation), and Sal-MSCs (cells pretreated with 0.75 μg/ml salidroside). Three days after MCAO, the anesthetized rats were fixed on a stereotaxic brain locator. For the MSC and Sal-MSC groups, 10 µl of cells (1 × 107 cells/ml) were engrafted at 3.6 mm toward the caudal side of the frontal fontanel and at 2 mm on the right side of the midline. The depth of the injection was 3 mm, and the rate of the injection was 1 µl/min. Rats of the vehicle group were transplanted with the same volume of PBS. The rats were euthanized 14 days after MCAO to obtain the brain tissue samples for 2,3,5-triphenyltetrazolium chloride (TTC) staining (n=3 per group), hematoxylin-eosin (HE) staining (n=3 per group), and immunofluorescence (n=6 per group). Behavioral testing (each time point; n=3 per group) was performed at 7, 14, and 21 days after MCAO.
Behavioral Test
Grip strength was measured at 7, 14, and 21 days after MCAO as previously described [18]. Briefly, each rat was held by its tail and gripped the tension bar of the instrument using their limbs. Once the grip was secured, the animal was slowly pulled away from the bar. A digital reading (in Newtons) of 3 successive trials was obtained for each rat, and then averaged for analysis.
TTC staining
To measure infarct volumes, rats were decapitated at 14 days after MCAO. Brains were cut into 2-mm-thick coronal sections and incubated in 2% TTC (Sigma, USA) solution for 30 min at 37°C in the dark. Infarct volumes were determined by the ImagePro Plus 6.0 software and expressed as the percentage of the total volume of the cerebral tissue.
Preparation of paraffin section
Brains from anesthetized rats were fixed by transcardial perfusion with saline, followed by perfusion and immersion in 4% paraformaldehyde. Then, brains were removed, post-fixed, dehydrated in graded alcohol, and embedded in paraffin. Coronal sections at 4 μm were used for HE staining and immunofluorescence.
HE staining
Paraffin sections were subjected to conventional dewaxing, hydration, hematoxylin staining, and hydrochloric acid ethanol differentiation. After rinsing with distilled water, the sections were stained with 1% eosin solution (BOSTER, China) for 1 min with subsequent dehydration in graded alcohol; the sections were dewaxed in xylene and mounted with neutral gum. The mounted slides were observed and imaged by microscope.
Immunofluorescence
CM-Dil-labeled MSCs were observed by fluorescence microscopy to detect the implanted cells in the host brain. Neuronal nuclear antigen (NeuN) and glial fibrillary acidic protein (GFAP) expression were assessed by evaluating the number of NeuN- and GFAP-positive cells in the ischemic boundary zone. Sections were dewaxed, hydrated, repaired by citric acid antigen, and incubated with 3% H2O2 for 10 min at room temperature. Then, sections were incubated with rabbit anti-NeuN antibody (1:5,000, Abcam) and rabbit anti-GFAP antibody (1:5,000, Abcam) overnight at 4°C and incubated with a secondary antibody at 37°C for 30 min. Whole sections were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma, USA). Ten fields of each section were randomly imaged at high magnification (400×) to count the CM-Dil-labeled MSCs NeuN- and GFAP-positive cells. Positive cells were counted by ImagePro Plus 6.0.
Statistical analysis
All experimental data are expressed as the mean ± standard deviation. The differences between multiple groups were analyzed by one-way analysis of variance (ANOVA). Comparisons between two groups were performed via the Dunnett's t-test. All were considered statistically significant for P<0.05.