Ethics
All animal experimental procedures were approved by the Institute of Animal Care and Use Committee at our hospital and performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, National Academy Press, Washington, DC, USA, revised 1996).
Isolation of Adipose-Derived Mesenchymal Stem Cells from Rat
The rats were anesthetized with inhalational isoflurane. Adipose tissue surrounding the epididymis was carefully dissected and excised. Then 200-300 μL of sterile saline was added to every 0.5 g of tissue to prevent dehydration. The tissue was cut into < 1 mm3 size pieces using a sharp, sterile surgical scissors. Sterile saline (37°C) was added to the homogenized adipose tissue in a ratio of 3:1 (saline: adipose tissue), followed by the addition of stock collagenase solution to a final concentration of 0.5 Units/mL. The tubes with the contents were placed and secured on a Thermaline shaker and incubated with constant agitation for 60 ± 15 min at 37°C. After 40 minutes of incubation, the content was triturated with a 25 mL pipette for 2-3 min. The cells obtained were placed back to the rocker for incubation. The contents of the flask were transferred to 50 mL tubes after digestion, followed by centrifugation at 600 g, for 5 minutes at room temperature. The fat layer and saline supernatant from the tube were poured out gently in one smooth motion or removed using vacuum suction. The cell pellet thus obtained was resuspended in 40 mL saline and then centrifuged again at 600 g for 5 minutes at room temperature. After being resuspended again in 5 mL saline, the cell suspension was filtered through a 100 μm filter into a 50 mL conical tube to which 2 mL of saline was added to rinse the remaining cells through the filter. The flow-through was pipetted to a 40 μm filter into a new 50 mL conical tube. The tubes were centrifuged for a third time at 600 g for 5 minutes at room temperature. The cells were resuspended in saline. An aliquot of cell suspension was then removed for cell culture in DMEM-low glucose medium contain 10% FBS for two weeks. Flow cytometric analysis was performed for identification of cellular characteristics after cell-labeling with appropriate antibodies 30 minutes before transplantation (Figure 1).
ADMSCs Labeling Before Autologous Transplantation
Thirty min prior to autologous transplantatting ADMSCs, CM-Dil (Vybrant™ Dil cell-labeling solution, Molecular Probes, Inc.) (50 μg/ml) was added to the culture medium. This highly lipophilic carbocyanine dye, which has properties of low cytotoxicity and high resistance to intercellular transfer, can be added directly to normal culture media to uniformly label suspended or attached culture cells for their visibility in a brain infarct area (BIA) due to its distinctive fluorescence.
Animal Model of Acute Ischemic Stoke
Pathogen-free, adult male Sprague-Dawley (SD) rats, weighing 300-350 g (Charles River Technology, BioLASCO Taiwan Co., Ltd., Taiwan) were utilized in this study. After adipose-derived mesenchymal stem cells (ADMSCs) were cultured for two weeks, acute stroke was induced in the animals. After exposure of the left common carotid artery (LCCA) through a transverse neck incision, a small incision was made on the LCCA through which a nylon filament (0.28 mm in diameter) was advanced into the distal left internal carotid artery for occlusion of left middle cerebral artery (LMCA) to induce brain infarction of its supplying region. Three hours after occlusion, the nylon filament was removed, followed by closure of the muscle and skin in layers.
In Vivo Treatment Protocol
Ten healthy rats served as normal controls (group 1). The rats with acute IS were divided into group 2 (acute IS treated with 1 mL intravenous physiological saline at 0, 12 and 24 h after IS induction, n = 15) and group 3 [acute IS plus intravenous ADMSCs (2.0 × 106 in 0.5 cc culture medium for each time) given at 0, 12 and 24 h after IS induction, n = 15). Five rats in groups 2 and 3 were utilized for determining the brain infarct size. The sensorimotor functional test (Corner test) was performed by blinded investigators for each rat on days 0, 1, 3, 7, 14 and 21 after acute IS induction as previously described [21].
Cellular Proliferation Test
To evaluate whether ADMSC treatment promotes cellular proliferation in the BIA, 5-bromodeoxyuridine (BrdU) was intravenously given in all three groups of animals on days 3, 5, 7, 9, and 12 after acute IS induction for labeling the proliferating cells.
Specimen Collection
Rats in groups 1, 2, and 3 were euthanized on day 21 after IS induction, and brain in each rat was rapidly removed and immersed in cold saline. For immunohistofluorescence (IHF) study, the brain tissue was rinsed with PBS, embedded in OCT compound (Tissue-Tek, Sakura, Netherlands) and snap-frozen in liquid nitrogen before being stored at -80°C. For immunohistochemical (IHC) staining, brain tissue was fixed in 4% formaldehyde and embedded in paraffin.
Measurement of Brain Infarct Area
To evaluate the impact of ADMSC treatment on brain infarction, coronal sections of the brain were obtained from five extra animals in group 2 and group 3 (n = 5 for each group) as 2 mm slices. Each cross section of brain tissue was then stained with 2% 3,5-Triphenyl-2H-Tetrazolium Chloride (TTC)(Alfa Aesar) for BIA analysis. Briefly, all brain sections were placed on a tray with a scaled vertical bar to which a digital camera was attached. The sections were photographed from directly above at a fixed height. The images obtained were then analyzed using Image Tool 3 (IT3) image analysis software (University of Texas, Health Science Center, San Antonio, UTHSCSA; Image Tool for Windows, Version 3.0, USA). Infarct area was observed as either whitish or pale yellowish regions. Infarct region was further confirmed by microscopic examination. The percentages of infarct area were then obtained by dividing the area with total cross-sectional area of the brain.
TUNEL Assay for Apoptotic Nuclei
For each rat, 6 sections of BIA were analyzed by an in situ Cell Death Detection Kit, AP (Roche) according to the manufacturer's guidelines. Three randomly chosen high-power fields (HPFs) (×400) were observed for terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling (TUNEL)-positive cells. The mean number of apoptotic nuclei per HPF for each animal was obtained by dividing the total number of cells with 18.
IHC Staining for Cellular Proliferation and Glial Fibrillary Acid Protein (GFAP)
Paraffin sections (5 μm thick) with BIA were obtained from each rat. To block the action of endogenous peroxidase, the sections were initially incubated with 3% hydrogen peroxide for 15 minutes, and then further processed using Beat Blocker Kit (invitrogen, #50-300) with immersion in solutions A and B for 30 minutes and 10 minutes at room temperature, respectively. Rabbit polyclonal antibody (1:500 dilution at 4°C overnight) against glial fibrillary acid protein (GFAP) (Dako) and monoclonal antibody (1:200 dilution at 4°C overnight) against 5-Bromo-2-DeoxyUridine (BrdU) (Sigma), were used as primary antibodies. The anti-rabbit HRP (Zymed) (1:3 dilution at room temperature for 10 minutes) for GFAP and anti-mouse HRP (Zymed) (1:3 dilution at room temperature for 10 minutes) were used as secondary antibodies, followed by application of SuperPicTure™ Polymer Detection Kit (Zymed) for 10 minutes at room temperature. Finally, the sections were counterstained with hematoxylin. For negative control experiments, primary antibodies were omitted.
Western Blot Analysis for CXCR4 and Stromal Cell-Derived Factor-1 in BIA
Equal amounts (60 μg) of protein extracts from BIA were loaded and separated by SDS-PAGE using 12-13% acrylamide gradients. Following electrophoresis, the separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences). Nonspecific proteins were blocked by incubating the membrane in blocking buffer (5% nonfat dry milk in T-TBS containing 0.05% Tween 20) overnight for CXCR4 and one hour for stromal cell-derived factor (SDF)-1, respectively. The membranes were incubated with the indicated primary antibodies (CXCR4, 1:1000, Abcam, Actin 1:10000, Chemicon; SDF-1, 1:1000, Cell Signaling) for one hour at room temperature for CXCR4 and overnight at 4°C for SDF-1, respectively. Horseradish peroxidase-conjugated anti-rabbit immunoglobulin IgG (1:2000, Cell Signaling) was applied as the secondary antibody for one hour for CXCR4 and 45 minutes for SDF-1 at room temperature. The washing procedure was repeated eight times within an hour, and immunoreactive bands were visualized by enhanced chemiluminescence (ECL) (Amersham Biosciences) and exposure to Biomax L film (Kodak). For quantification, digitized ECL signals were analyzed using Labwork UVP software.
Protocol for RNA Extraction
Lysis/binding buffer (High Pure RNA Tissue Kit, Roche, Germany) 400 μL and an appropriate amount of frozen brain tissue were added to a nuclease-free 1.5 mL microcentrifuge tube, followed by disruption and homogenization of the tissue by using a rotor-stator homogenizer (Roche).
For each isolation, 90 μL DNase incubation buffer was pipetted into a sterile 1.5 mL reaction tube, 10 mL DNase I working solution was then added, mixed and incubated for 15 minutes at 25°C. Washing buffer I 500 μL was then added to the upper reservoir of the filter tube, which was then centrifuged for 15 seconds at 8,000 g. Washing buffer II 300 μL was added to the upper reservoir of the filter tube, which was centrifuged for 2 minutes full-speed at approximately 13,000 g. Elution Buffer 100 μL was added to the upper reservoir of the filter tube; the tube assembly was then centrifuged for one minute at 8,000 g resulting in eluted RNA in the microcentrifuge tube.
Real-Time Quantitative PCR Analysis
Real-time polymerase chain reaction (RT-PCR) was conducted using LightCycler TaqMan Master (Roche, Germany) in a single capillary tube according to the manufacturer's guidelines for individual component concentrations. Forward and reverse primers were each designed based on individual exons of the target gene sequence to avoid amplifying genomic DNA.
During PCR, the probe was hybridized to its complementary single-strand DNA sequence within the PCR target. As amplification occurred, the probe was degraded due to the exonuclease activity of Taq DNA polymerase, thereby separating the quencher from reporter dye during extension. During the entire amplification cycle, light emission increased exponentially. A positive result was determined by identifying the threshold cycle value at which reporter dye emission appeared above background.
Immunohistofluorescence (IHF) analysis for CXCR4, SDF-1, Doublecortin, and von Willebrand Factor (vWF)
Serial cryosections (7 μm thick) with an average distance of 5 μm apart were collected from the BIA. The sections were fixed in acetone for 15 minutes at -20°C. For reducing the background, 200 μL of signal enhancer was utilized for blocking non-specific signals at room temperature for 30 minutes. IHF staining was performed using primary antibody (rabbit polyclonal antibody 1:200 dilution, at 4°C, overnight) (Santa Cruz) for CXCR4, followed by the addition of anti-rabbit Alexa Fluor 488 FITC (Molecular Probes) secondary antibody (1:200 dilution at room temperature for 30 minutes). Additionally, rabbit polyclonal antibody (1:500 dilution at 4°C overnight) (Santa Cruz) was used as primary antibody for SDF-1, followed by the addition of anti-rabbit Alexa Fluor 594 Rodamin (Molecular Probes) secondary antibody (1:200 dilution at room temperature for 30 minutes). Moreover, goat polyclonal antibody (1:50 dilution, at 4°C overnight) (Santa Cruz) was used as primary antibody to recognize doublecortin, followed by anti-goat Alexa Fluor 568 Rodamin (Molecular Probes) secondary antibody (1:200 dilution at room temperature for 30 minutes). Furthermore, rabbit polyclonal antibody (1:200 dilution at 4°C overnight) (Chemicon) was used as primary antibody against vWF, followed by anti-rabbit Alexa Fluor 488 FITC (Molecular Probes) secondary antibody (1:200 dilution at room temperature for 30 minutes). For negative control experiments, the primary antibodies were omitted. The sections were counterstained with 4', 6-Diamidino-2-phenylindole (DAPI) (dilution 1/500) (Sigma) to identify cellular nuclei that represented the cell number.
Oxidative Stress of BIA
The Oxyblot Oxidized Protein Detection Kit was purchased from Chemicon (S7150). The 2,4-dinitrophenylhydrazine (DNPH) derivatization was carried out on 6 μg of protein for 15 minutes according to manufacturer's instructions. One-dimensional electrophoresis was carried out on 12% SDS/polyacrylamide gel after DNPH derivatization. Proteins were transferred to nitrocellulose membranes which were then incubated in the primary antibody solution (anti-DNP 1: 150) for 2 hours, followed by incubation with second antibody solution (1:300) for one hour at room temperature. The washing procedure was repeated eight times within 40 minutes. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) which was then exposed to Biomax L film (Kodak). For quantification, ECL signals were digitized using Labwork software (UVP). On each gel, a standard control was loaded.
Small Vessel Density in BIA
IHC staining of small blood vessels (i.e. diameters ≤ 15 mm) was performed with anti-α-SMA (1:400) as primary antibody at room temperature for one hour, followed by washing with PBS thrice. The anti-mouse HRP-conjugated secondary antibody was then added and incubated for 10 minutes, followed by washing with PBS thrice. Then 3,3' diaminobenzidine (DAB) (0.7 gm/tablet) (Sigma) was added and incubated for one minute, followed by washing with PBS thrice. Finally, following hematoxylin treatment for one minute as a counter stain for nuclei, the sections were washed twice. Three coronal sections of the brain were analyzed in each rat. For quantification, three randomly selected HPFs (200×) were analyzed in each section. The mean number of small vessel per HPF for each animal was then determined by summation of all numbers divided by 9.
Statistical Analysis
Data were expressed as mean values (mean ± SD). The significance of differences between two groups was evaluated with t-test. The significance of differences among three groups was evaluated using analysis of variance followed by Bonferroni multiple-comparison post hoc test. Statistical analyses were performed using SAS statistical software for Windows version 8.2 (SAS institute, Cary, NC). A probability value < 0.05 was considered statistically significant.