Histological and Immunohistochemical Assessment

8.1. TTC Staining and Quantitative Analysis of Infarct Volume

The brains were removed immediately and sectioned coronally into six slices (2 mm thick) using a rodent brain matrix (Harvard Instrument Inc., South Natick, MA). Brain slices were placed in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC; Sigma Chemical, St. Louis, MO) in the dark and incubated at 37°C for 30 min. The brain slices were then removed from the incubator. The cross-sectional area of infarction in each brain slice was examined under a dissection microscope and measured using image analysis software (TINA 2.0; Raytest, Straubenhardt, Germany). The total infarct volume for each brain was calculated by summing the infarcted area of all brain slices.

paraformaldehyde solution. The brains were removed quickly and kept in 4%

paraformaldehyde solution overnight at 4°C, embedded in 30% sucrose solution until they sank, and frozen sectioned on a slidingmicrotome in 30-µm-thick coronal sections. To count BrdU-positive cells in the left subventricular zone (SVZ) and the ischemic boundary zone (IBZ), systematic random sampling was conducted, as previously described (Prockop et al., 1997). Every sixth section in a series of 60-µm-thick coronal sections between bregma levels +1.0 and –1.0 mm (total of six sections per brain) were collected to quantify BrdU labeling.

I counted BrdU-positive cells to evaluate the degree of neurogenesis. To do so, DNA was first denatured by incubating brain sections in 2 N HCl at 37°C for 1 h. The sections were then rinsed with Tris buffer and treated with 0.3% H₂O₂ to block endogenous peroxidase. The sections were blocked with 10% normal horse serum and 0.1% Triton X-100 in PBS for 30 min at room temperature and incubated with a mouse monoclonal antibody (mAb) against BrdU (1:200; Roche) overnight at 4°C. The sections were then incubated with horse anti-mouse IgG for 1 h at room temperature and with an avidin–biotin–peroxidase complex (Vectastain ABC kit; Vector Laboratories, Burlingame,CA) for 1 h at room temperature and developed in diaminobenzidine (DAB) substrate-chromogen (DAB kit;

DakoCytomation, Glostrup, Denmark). The tissues were rinsed in 0.1 M PBS to stop the DAB reaction. The labeled tissue sections were then mounted on gelatin-coated slides and analyzed under a bright-field microscope (E600; Nikon, Tokyo, Japan).

8. 3. Stereological Cell Counts

To determine the number of BrdU-labeled cells in the SVZ and IBZ of the ipsilateral

hemisphere, every 6^th section (each 60 µm) between bregma levels +1.0 and –1.0 mm was selected (total of five sections per brain). The unbiased stereological estimate of the total number of BrdU-immunopositive cells inthe SVZ and IBZ was made using the optical fractionator technique (Plane et al., 2004). This sampling technique is not affected by tissue volume changes and does not require reference volume determinations(West et al., 1991).

Sampling was performed with the Computer-Assisted Stereological Toolbox (CAST) system (version 2.3.1.5, Olympus Denmark A/S, Ballerup, Denmark), using an Olympus BX51 microscope. The SVZ and IBZ were delineated using a 1.25× objective, which generated counting areas of 84.62 × 84.62 µm. A counting frame (3,580 µm²) was placed randomly on the first counting area and moved through all the counting areas systemically until the entire delineated area was sampled. The sampling frequency was chosen so that 100–200 BrdU-positive cells were counted in each specimen in the ipsilateral hemisphere. Actual counting was performed using a 100× oil objective lens. Guard volumes (4 µm from the top and 4–6 µm from the bottom of the section) were excluded from both surfaces to avoid the problem of lost caps, and only the profiles that came into focus within the counting volume (with a depth of 20 µm) were counted. The total number of BrdU-positive cells was estimated using the optical fractionator formula (West et al., 1991).

For double-immunofluorescence staining, the tissues were washed three times with PBS and nonspecific binding was blocked with 10% normal horse serum. Then, the tissues were treated overnight at 4°C with monoclonal antibody specific for human nuclei matrix antigen (NuMA; Oncogen, Seattle, WA) diluted 1:100 in PBS. Following sequential incubation with anti-mouse Alexa 594 (1:400; Molecular Probes, Eugene, OR), secondary antibody was bound to the first antibody to NuMA. Cells derived from hMSCs were identified using morphologic criteria and immunohistochemical staining with NuMA (present in the donor cells, but not present in the parenchymal cells).

To visualize the cellular colocalization of NuMA and cell-type-specific markers in the same cells, each coronal slide was treated with the first primary antibody to NuMA, as described above, and then incubated with glial fibrillary acidic protein (GFAP for astrocytes;

Sigma, St. Louis, MO), the neurofilament subunit (NF-L for neurons; Chemicon, Temecula, CA), and doublecortin (DCX C-18 for migrating neurons; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C and then incubated with anti-rabbit Alexa 488 (1:400; Molecular Probes), anti-mouse Alexa 488 (1:400; Molecular Probes), or anti-goat FITC (1:200; Vector Laboratories) antibody for 1 h at room temperature. The sections were washed in PBS, rinsed with double-distilled water, and mounted on slides using an anti-fade mounting medium (Permafluor; Molecular Probes).

To determine whether BrdU-labeled cells differentiate into neuronal or astroglial cells, sections were treated with 2 N HCl and rinsed as above, blocked with 10% normal horse or goat serum and 0.1% Triton X-100 in PBS for 30 min at room temperature, and incubated with rat monoclonal anti-BrdU (1:100; BD Biosciences, San Jose, CA) overnight at 4°C.

After rinsing with primary antibodies, the sections were incubated for 1 h with cy3-conjugated goat anti-rat IgG (1:400; Abcom, Cambridge, UK). The sections were then incubated with NF-L, neuronal nuclear protein (NeuN for neuron; Chemicon), DCX, and GFAP overnight at 4°C, and incubated with anti-rabbit Alexa 488 (1:400), anti-mouse Alexa 488 (1:400), or anti-goat FITC (1:200) antibody for 1 h at room temperature. The sections were washed in PBS, rinsed with double-distilled water, and mounted on slides using an anti-fade mounting medium (Permafluor, Molecular Probes). The specimens were examined on a Zeiss LSM510 confocal imaging system (Carl Zeiss).

To assess what percentage of newborn cells differentiated into a neuronal phenotype after hMSC treatment, the double-positive percentage was calculated from the number of double-positive cells and the total number of BrdU-positive cells. The mean values were calculated from eight slides from four rats, each slide randomly containing eight fields from the hemisphere.