Isolation of mesenchymal stem cells
Cultures were made of BM-MSC obtained from the tibia and femur of adult female Sprague-Dawley (250-300 g) rats that had been sacrificed by cardiac injection of potassium chloride (0.5 ml) as described previously . First, both bones were placed in alcohol for 10 minutes and then in Hank's (1X) balanced salt solution (HBSS, Gibco, Paisley, UK) for 30 minutes. The bones were cut and bone marrow removed, 10 ml of this tissue was placed in 50 ml of DMEM 1X solution (DMEM Glu/Pyr, Gibco) with 75 μl penicillin/streptomycin (Sigma-Aldrich, St Louis, MO, USA) and 20% FBS (PAA Laboratories, GmbH, Pasching, Austria). Next, cells were washed and centrifuged twice at 390 g for 8 minutes at room temperature. The cells were placed in 75-cm2 flasks (Nunc, Roskilde, Denmark)) and incubated at 37°C in 5% CO2 for 3 to 4 weeks. The culture medium was replaced approximately every 3 days. When cells reached 80 to 90% confluence, they were trypsinized using Trypsin-EDTA 0. 05% (Gibco) and expanded in another 75-cm2 flask. On the third pass they were trypsinized and counted before being administered to the experimental animals.
Lipoaspirates from adult female Sprague-Dawley (250 to 300 g) were washed with sterile PBS and digested with an equal volume of 0.075% type I collagenase (Sigma-Aldrich,). The filtered cells were centrifuged at 390 g for 10 minutes and contaminating erythrocytes were removed to isolate the stromal vascular fraction (SVF). On the third pass they were trypsinized and counted before being administered to the experimental animals.
Characterization of MSC
At the time the cells were obtained, the cultures were characterized to confirm the presence or absence of MSC surface markers using the flow cytometric technique and analyzed with fluorescence-activated cell sorting (FACS). The cells were incubated for 20 minutes at 4°C in the dark with the following antibodies: CD90-fluorescein isothiocyanate (FITC) (AbD Serotec, Oxford, UK), CD29-Phycoerythrin (PE) (AbD Serotec), CD45-PE (AbD Serotec) and CD11b-PE (AbD Serotec). Matched isotype controls were purchased from Biolegend (Biolegend, San Diego, CA, USA). At least 1 × 104 cells per sample were acquired and analyzed.
Subjects were adult male Sprague-Dawley rats, with an average body weight range of 250 to 320 g (Harlan Iberica SL, Barcelona, Spain). Animals were housed with free access to food and water at a room temperature of 21 ± SD 2°C, relative humidity of 45 ± 15% and a light/dark cycle of 12 h (7:00 to 19:00).
The animals were randomly assigned to one of four experimental groups with 10 animals in each study group: group 1, the sham-operated group, underwent surgery without infarct and received a saline solution via the femoral vein; group 2, the iInfarct group, underwent surgery with permanent middle cerebral artery occlusion (pMCAO) and received the saline infusion via the femoral vein; group 3, the BM-MSC group, underwent pMCAO surgery and received a BM-MSC infusion via the femoral vein; and group 4, the AD-MSC group, underwent pMCAO surgery and received an AD-MSC infusion via the femoral vein.
Anesthesia was induced by intraperitoneal injection of a solution of ketamine (25 mg/kg), diazepam (2 mg/kg), and atropine (0.1 mg/kg) at a dose of 2.5 ml/kg. Analgesia was provided by meloxicam 2 mg/kg by a subcutaneous route. A small craniectomy was made above the rhinal fissure over the branch of the right middle cerebral artery (MCA). The MCA branch was permanently ligated just before its bifurcation into the frontal and parietal branches with a 9-0 suture. Both common carotid arteries were then occluded for 60 minutes as previously described .
In all animals, the femoral artery was cannulated during surgery and ischemia, to allow continuous monitoring of physiological parameters (glycemia, blood gases and blood pressure) (Monitor Omicron ALTEA RGB medical devices, Madrid, Spain). Cranial and body temperature were also monitored and maintained at 36.5 ± 0.5°C.
Intravenous injections of 2 × 106 MSC in 650 μl saline were administered over 4 minutes through the femoral vein. Infarct animals underwent cerebral ischemia as in the treated animals but received only a saline infusion through the femoral vein. Sham-operated animals received the saline infusion through the femoral vein but did not undergo cerebral ischemia. The sham-operated and infarct groups both received a single 650 μl saline infusion without MSCs over 4 minutes. Either the saline or MSC solution was administered in the acute phase 30 minutes after common carotid artery reperfusion. The route, dose and timing of administration have been used in a previous study .
In vivo analyses
Functional evaluation scales
In all animals functional evaluation scales were performed at baseline and at 24 h and 14 d after surgery. All rats were evaluated using a variant of the Rogers scale [8, 19–21] and the rotarod test. The Rogers scale scores functional status as follows: no deficit (0); failure to extend left forepaw (1); decreased grip of the left forelimb when the tail is pulled (2); spontaneous movement in all directions, contralateral circling if pulled (3); circling or walking to the left (4); movement only when stimulated (5); unresponsive to stimulation (6); and dead (7). The rotarod test was used to evaluate the motor performance of the rats. Beginning three days before pMCAO, rats were trained on an accelerating (4 to 40 rpm) rotarod. All animals received a 3-day training program consisting of three sessions per day, and the time each animal remained on the rotarod was measured. Before surgery on the experimental day, the time spent moving on the rotarod without falling was measured twice per animal with a 15-minutes interval between each trial. The mean of the two trials was calculated for each rat .
Migration and implantation of stem cells by magnetic resonance imaging (MRI)
MSC were magnetically labeled using Endorem™ (superparamagnetic iron oxide). (Guerbet, Roissy CdG Cedex, France). Both migration and implantation of the stem cells were analyzed at 24 hours and 14 d by MRI with T2 maps (flash sequence). Endorem™(Guerbet)-labeled MSC were transplanted into five animals in each group.
Measurement of volume of ischemic lesion by MRI
Lesion volume was analyzed at 24 h and 14 days after surgery by MRI (Bruker Pharmascan, Ettlingen, Germany), (7 Tesla horizontal bore magnets) using T2 maps (RARE 8 T2, 180° flip angle, three averages). Ten contiguous coronal slices (thickness, 1 mm) were acquired with a field of view of 35 × 35 mm and a matrix size of 256 × 256 (repetition time (TR) 3000 ms, echo time (TE) 29.5 ms, imaging time 25.5 minutes, three averages). All images were processed using the J 1.42 Image program (NIH software, Bethesda, MD, USA). After contrast adjustment, the contours of the hemispheres were traced manually on each slice. The infarct volumes were estimated by integrating the partial measurements derived from the cross-sectional areas and the distance between sections. To correct for the brain edema effect, lesion volume was determined by an indirect method:
Infarct area = (Area of the intact contralateral hemisphere) - (Area of the intact ipsilateral hemisphere) .
Then lesion volume was expressed as a percentage of the intact contralateral hemispheric volume.
Migration and implantation of stem cells by DiI
MSC were labeled with DiI (Celltracker CM-DiI, Molecular Probes ™, Eugene, Oregon, USA) prior to administration and then, migration and implantation were analyzed at 14 d post-administration using immunofluorescence. The DiI (Molecular Probes ™)-labeled MSC were administered into five animals in each treatment group.
Measurement of volume of ischemic lesion by H&E
Lesion size was estimated with H&E staining of brain sections at 14 d. Infarction volume was expressed as the percentage of brain tissue affected by ischemia in the right hemisphere as evaluated on 10-μm-thick sections. Brains were sectioned at the optic chiasma and at the infundibular stalk. The resultant brain block from between these two cuts was placed in 4% paraformaldehyde for 24 h and 30% sacarose PBS buffer for 3 days. Optimal cutting temperature (OCT)-embedded samples were coronally sectioned in 10-μm-thick slices. Every twentieth slice, that is, a total of four of these slices (numbers 1, 21, 41 and 61), separated from each other by 100 μm, were stained with H&E, which reveals an ischemic area as a well-defined pale region. A digitized image was made of these slices (Epson Perfection 1260 scanner, Suwa, Nagano, Japan) and used to automatically measure the ischemic area (Image Pro plus 4.0, Media Cybernetics, Rockville, MD, USA) [8, 20, 24]. Lesion volume was determined using the method described in MRI measurement of volume of ischemic lesion.
Apoptotic cell death was detected by biotin-dUTP nick end-labeling mediated by terminal deoxynucleotidyl transferase (TUNEL) staining, using TdT-FragEL DNA Fragmentation Detection Kit, Oncogene Research Products, San Diego, CA, USA) following the methodology indicated by the manufacturer. We chose a single rostral-caudal coronal section per animal and counted the number of apoptotic cells in the peri-infarct zone using a 40× objective on the optic microscope (Olympus, BX41, Olympus Corporation, Tokyo, Japan) and image analysis software (Image-Pro Plus 4.1, Media Cybernetics, Rockville, MD, USA). Cells undergoing apoptosis were identified based on their nuclear morphology and dark color [8, 20].
All animals were given 50 mg/kg of daily intraperitoneal BrdU (Sigma-Aldrich) on days 4 to 7 after ischemia. This administration protocol was based on previous reports that proliferation peaked 4 to 10 days after injury [25, 26]. Animals were sacrificed 14 d after surgery by transcardial perfusion and decapitation. Their brains were fixed and stored at 4°C and the following day the tissue was placed in cryoprotectant solution at -80°C. Serial coronal sections were cut at 10 μm on a cryostat (LEICA CM1950, Leica Microsystems, Heilderbeg, Germany) and later studied by immunohistochemistry for cellular proliferation. Brain sections were treated with BrdU In-Situ Detection kit (BD Biosciences, Franklin Lakes, NJ, USA).
The sections were studied using different immunofluorescent antibodies as follows: the neuronal markers, neuronal nuclei (NeuN) (monoclonal antibody diluted 1:100, Millipore, Billerica, MA, USA) and neurofilament (NF) (monoclonal antibody diluted 1:100, DAKO, Denmark A/S, Glostrup, Denmark); the astrocyte marker, glial fiibrillary acid protein (GFAP) (monoclonal antibody diluted 1:400, Chemicon, Temecula, CA, USA); the vascular endothelial growth factor (VEGF) marker (polyclonal antibody diluted 1:500, Millipore); the oligodendrocyte (Olig-2) marker (polyclonal antibody diluted 1:500, Millipore); the synaptogenesis marker, synaptophysin (monoclonal antibody diluted 1:200, Sigma-Aldrich); and the brain-derived neurotrophic factor (BDNF) (polyclonal antibody diluted 1:1000, Millipore), followed by goat anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 594 (1: 750, Molecular Probes, Invitrogen, Barcelona, Spain). Also, we used BrdU (monoclonal antibody diluted 1:50, DAKO) followed by goat anti-mouse Alexa Fluor 594 as a proliferation marker. All sections were mounted with H-1200 VectaShield mounting medium for fluorescence with diamidino-2-phenylindole (DAPI, Vector, Atom, Alicante, Spain). Samples were examined using a LEICA TCS SPE spectral confocal microscope (Leica Microsystems, Heidelberg, Germany) and the confocal images were analyzed using LEICA software LAS AF, version 2.0.1 Build 2043. The images were acquired as a confocal maximum projection.
Proteins were isolated from peri-infarct tissue and their concentrations determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Twenty micrograms of protein were loaded onto 10% acrylamide SDS-gels. Following electrophoresis at 100 V for 1 h, protein was transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked in 5% fat-free dry milk dissolved in Tris-buffered saline pH 8.0 (TBS) plus 0.1% Tween-20 (TBS-T) for 1 h and probed overnight at 4°C with the following antibodies at the designated dilutions: NF (monoclonal antibody diluted 1:100, DAKO); GFAP (monoclonal antibody diluted 1:400, Chemicon); VEGF (polyclonal antibody diluted 1:500, Millipore); Olig-2 (polyclonal antibody diluted 1:500, Millipore); synaptophysin (monoclonal antibody diluted 1:200, Sigma-Aldrich); BDNF (polyclonal antibody diluted 1:1000, Millipore) and β-actin (monoclonal antibody diluted 1:400, Sigma-Aldrich), which was used as a control protein. After rinsing with 0.5% TBS-T solution, the membranes were incubated with the secondary antibody, a donkey anti-rabbit and anti-mouse antibody conjugated with horseradish peroxidase (HRP, Chemicon) for 1 h at room temperature. Signals were detected by enhanced chemiluminescence (ECL, GE, Healthcare Europe GmbH, Freiburg, Germany) before exposure on radiographic film. The density of stained bands was scanned and quantified by the Scion Image and 1-D Manager Version 2.1 (Scion Corporation, Frederick, Maryland, USA). To reduce differences between animals, at least three western blots were performed for each time point and animal. In addition, at least two or three repeated samples were always included in every set of experimental samples as internal standards.
Quantitative data are shown as mean values ± SD. The Kruskal-Wallis test followed by the Mann-Whitney test were used to compare the functional evaluation score, lesion size, cell death and number of BrdU-positive cells, while VEGF, BDNF, SYP, Olig-2, NF and GFAP levels were compared between animals receiving MSC from either cell source among the various groups. Values of P < 0.05 were considered significant at a 95% CI; we used statistical software SPSS 16 for Windows for analysis.