All SPF Sprague-Dawley (SD) rats were obtained from the Laboratory Animal Center of Southwest Medical University. The rats were housed at room temperature (23±2°C) in a 12 h/12 h light/dark cycle with free access to food and water. Experiments abided by all relevant ethical regulations and were approved by the Animal Care and Use Committee at the Southwest Medical University. Animal studies were conducted on the adult SD rats with weighing 220-250 g and aged 8-10 weeks at experimental onset. The animals were separated into sham operation (SO) group and intracerebral hemorrhage (ICH) group, and the latter were randomly subdivided into model control (MC) group, MSCs transplantation (MSCs) group, EA stimulation(EA) group and MSCs transplantation combined with EA stimulation (MSCs+EA) group.
ICH Rat Model
ICH rat model was established as previously described by injecting collagenase and heparin . Briefly, SD rat was anesthetized with 1% pentobarbital sodium (40mg/kg) via intraperitoneal administration, and then placed on a stereotaxic apparatus (Benchmark, myNeurolab.com, USA). The scalp was incised longitudinally with 1 cm long in the midline, and a 1 mm burr hole was drilled on the right cranial bone at the point that was 0.2 mm anterior and 3 mm lateral to bregma. A sterile 5-μl Hamilton syringe was then inserted into a point that was 6 mm ventral to the hole, and collagenase I (2μl, 0.125U/μl, Sigma) and heparin (1μl, 2U/μl) were slowly injected into the right striatum of rat. Then the needle was removed, and the burr hole was sealed with bone wax and the scalp was sutured. After operation, all rats were allowed free access to food and water. For the SO group, the same procedure was carried out in the rats without injection of collagenase I and heparin.
MSCs labelled with green fluorescent protein (GFP) (No. RASMX-01101, Cyagen Biosciences, Inc. Suzhou, People’s Republic of China; Inspection report seen in Supplementary Fig. 1) were recovered from liquid nitrogen and cultured by using alpha-minimum essential medium (α-MEM) (Hyclone) supplemented with 10% FBS (Hyclone), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco) in an incubator (37°C, 5%CO2). When the 3-5 generation of MSCs reached 80% confluence, the medium was substituted with preinduction solution comprised of α-MEM with 10% FBS and 1 mmol/L β-mercaptoethanol (β-ME) for 24 hours, then with neuronal induction solution composed of α-MEM with 2% dimethylsulfoxide (DMSO), 1 mmol/L β-ME and 1μmol/L all-trans retinoic acid (RA) (Sigma) for 6 hours. The cells were collected and the cell concentration was adjusted to 2.5 ×107 cells/ml.
At 2 days following ICH, rats of MSCs group and MSCs+EA group were anesthetized and placed on a stereotaxic apparatus again. The suture was disassembled and a sterile microsyringe with cell suspension was inserted into the point (coordinates: 0.2 mm anterior, 6 mm ventral, 3 mm lateral to the bregma) via the burr hole described above. Then, 5×105 cells in the total 20 µL saline were slowly injected into the right striatum. After cell transplantation, the hole was filled with bone wax and the skin incision was sutured again. The animals survived for 2 weeks after cell implantation.
The EA stimulation was performed according to the methodological standards as previously described  and the Baihui (GV20, located at the intersection of the median line of the head and the line connecting the apexes of the two auricles) and Dazhui (GV14, located at the posterior midline of the neck and between the spinous process of the seventh cervical vertebra and that of the first thoracic vertebra) acupoints were selected for EA stimulation. After cleaning the skin with alcohol swabs, the researcher inserted a sterile acupuncture needle into GV20 to the depths of 2 mm obliquely, and another needle into GV14 to the depths of 5mm vertically to the skin. Then both of the needles were connected to EA stimulator (HM6805, People’s Republic of China), and the rats were stimulated with continuous wave, intensity of 1 mA, frequency of 3 Hz and stimulation duration of 10 min. Rats of EA group and MSCs+EA group received EA stimulation once a day at 2 days following ICH, and fourteen days in succession until the rats were sacrificed. Rats of MC group were not treated with EA and MSCs implantation.
Neurological behavioral score
At 2 h, 1 d, 2 d following the induction of ICH and 14 d after treatment, modified Neurological Severity Scores (mNSS) was used to evaluate the neurologic deficits of each rat by an investigator who was blind to the experiment design. The score is composed of sensory, motor, reflex, and balance tests. The degree of neurological deficits is graded on a scale of 0 to 18 (normal score, 0; maximal deficit score, 18). Rats with higher score showed more serious injury. Only rats with score of more than 8 at 2h after ICH induction were used in the current study. The reduction of mNSS score of each rat is the difference in scores between 14 days after treatment and 1 day after ICH.
The TreadScan Gait System (Clever System Inc, USA), consists of a treadmill device and a high-speed digital video camera, was used to obtain and evaluate the gait of the ICH rats at 14 days after treatment following the provided protocol and previously described . Rats were tested on the treadmill at the speed of 8 cm/s for 20 s sessions, and 2000 digital camera frames were captured. Gait performance was recorded and assessed using the TreadScan software. The gait parameters measured in this study are as follows: stride length, the run speed, print area, foot and pressure.
Positron-emission tomography/computed tomography (PET/CT) imaging was performed by micro PET/CT (Siemense, German). 18F- fluorodeoxyglucose (18F-FDG) was prepared at the Department of Nuclear Medicine, Affiliated Hospital of Southwest Medical University. After overnight fast, the rats were injected with approximately 0.8 mCi/kg of 18F-FDG through tail vein, then returned to their home cage to allow the uptake of 18F-FDG. About 30 min later, the animals were anesthetized with 1% pentobarbital sodium (40mg/kg, i.p.) and placed in the micro PET/CT scanner. Subsequently, PET scan and CT scan were performed for head imaging (Scan duration 15 min). The hemorrhagic foci volume and its glucose uptake were detected by analyzing the PET and CT imaging of rats.
After the neurologic evaluation and PET/CT scans at 14 days post-treatment, the rats were euthanized with an overdose anesthetic drug and perfused transcardially with 0.9% saline and 4% paraformaldehyde (PFA), consecutively. Brains were taken out and submerged in PFA for 1 day, and dehydrated in 15%, and 30% sucrose solution until the brain sank, then embedded in Tissue-Tek® O.C.T.Compound (Sakura Finetek). The brains were cut into 7-µm coronal sections on a freezing microtome (Leica CM1850) and stored at -20°C. Sections were selected from a region spanning from -0.4 mm to +1.8 mm with respect to the bregma, and stained with hematoxylin and eosin (HE) for pathomorphological observation.
Assessing the morphological change of mitochondria using TEM
Dissected caudoputamen samples (the peripheral area of hemorrhagic foci, taking this point located 1.7 mm lateral to the injection position as the center) were separated into 1 mm3 pieces, then fixed with 3% glutaraldehyde at 4°C. The pieces were post-fixed in 1% osmium tetroxide for 2 h, dehydrated in a gradient series of acetone, infiltrated with propylene epoxide, and finally embedded in Epon 618. Ultra-thin sections (40 nm) were cut with ultramicrotome, collected on copper grids, stained with uranium acetate and lead citrate. Four sections were selected from each group, and seven visual fields in each section were examined with TEM (JEM-1400/1011, Japan). The number of mitochondria was counted in an area of 100 µm2, and the rate of damaged mitochondrial were calculated with the following formula: rate = (number of damaged mitochondria /total number of mitochondria) × 100%.
Enzyme-Linked Immunosorbent Assay (ELISA)
After anesthesia, blood of rats was collected for preparation of serum and plasma. Brain damage markers and oxidative stress factor contents in serum or plasma were determined by ELISA. According to the manufacturer’s instructions, the expression levels of brain damage markers (MBP, NSE, S100B) and oxidative stress factors (MDA and SOD) in the rat serum/plasma were detected by ELISA kits from Novus Biologicals (MBP) and Nanjing Jiancheng Corp. ( NSE, S100B, MDA, SOD). The experimental process of different ELISA kits is not exactly the same. Briefly (taking the MBP kit as an example), 100 µl standard working solution and l00 µl diluted sample were added to the corresponding plate wells, and incubated for 90 min at 37°C . Remove the liquid out of each well and immediately add 100 μL of biotinylated detection Ab working solution to each well, and incubate for 1 hour at 37°C. After the plate was washed, l00 μl HRP conjugate working solution was added to each well and incubated for 30 minutes at 37°C. After the plate was washed, 90 μl substrate reagent was added to each well and for 15 min at 37°C. The reaction was terminated by adding 50 µl stop solution. The absorbance values were detected at 450 nm on a Microplate Reader (Thermo, USA). The standard curve was drawn and the corresponding concentration of the sample was calculated according to the curve equation.
Immunohistochemistry was performed as follows. Frozen sections were soaked in phosphate buffer saline (PBS) for 10 min and then incubated in 0.3% Triton-X 100 for 10 min. Next, 3% hydrogen superoxide and 10% normal goat serum were used to quench endogenous peroxidase activity and block non-specific binding respectively. Following removal of blocking buffer, the sections were incubated with monoclonal anti-rat Bax (1:200, Santa cruz) , Bcl-2 (1:200, Santa cruz), Arg-1 (1:100, CST) and iNOS (1:100, Bioworld) at 4˚C overnight.Then corresponding biotinylated secondary antibody solution and avidin-biotin-peroxidase reagent were added to the sections, and diaminobenzidine was used for colorization. Finally, the samples were counterstained with hematoxylin. Negative controls were identically processed, except that the primary antibody was omitted.
For immunofluorescence, brain slices were washed with PBS for 10 minutes and permeabilized in 0.1% Triton X-100 solution for 20 min at room temperature, then blocked in 10% normal goat serum for 30 min. The samples were incubated with monoclonal anti-rat MAP2 (1:200, Santa cruz) at 4˚C overnight and Alexa Fluor 594-conjugated goat anti-mouse IgG (1:500, Invitrogen) at room temperature for 60 min. Finally, slices were covered with fluorescence mounting medium (Dako).
TdT-mediated dUTP nick-end labeling (TUNEL) staining
Frozen sections were rinsed three times in PBS and then permeabilized with 0.1% Triton X-100. After washing two times in PBS, the sections were treated with TUNEL reaction mixture (Roche) for 60 min at 37˚C in the dark. After washing three times in PBS, the sections were incubated with 4',6-diamidino-2-phenylindole (DAPI) for 5 minutes at room temperature, then covered with fluorescence mounting medium. Negative controls were identically processed, except that the TUNEL reaction mixture was omitted.
Rats were anesthetized with 1% pentobarbital sodium and decapitated, then the ipsilateral striatum was immediately taken and quickly frozen in liquid nitrogen until homogenization. Total proteins were extracted from brain samples using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime) added with phenylmethanesulfonyl fluoride (PMSF) (Beyotime). The protein concentration of the lysates was quantified by the bicinchoninic acid (BCA) protein assay kit (Beyotime). Equal amounts of total protein (10 μg) from different samples were resolved by 10-12 % sodium dodecyl sulfate polyacrylamide gel by electrophoresis (SDS-PAGE), then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore) by trans-blot system (Bio-Rad) for detection. The membranes were incubated in blocking buffer (tris-buffer saline-tween, 5% dried milk) at room temperature for 30 min, following by primary antibodies (BDNF, NGF, COX4, OGDH, PDH-E1α, Bax, Bcl-2, GAPDH and HSP70) at 4˚C overnight, then secondary horseradish peroxidase (HRP)-labeled goat anti-rabbit or anti-mouse IgG (Bio-Rad) at room temperature for 60 min. The targeted protein bands were visualized with horseradish peroxidase chromogenic Kit (Thermo Fisher), and the density of each band was quantified with an image analysis software (Quantity one). HSP70 or GAPDH was the internal control.
The statistical analyses were performed with GraphPad Prism 6.0 (San Diego, CA, USA). All data are presented as mean ± s.e.m. Statistical significance is determined using unpaired Student’s t-test between two groups and one-way analysis of variance test for multiple comparisons when comparing multiple groups. P value < 0.05 was considered to be statistically significant.