Sublethal Transient Global Ischemia
All animal experiments and surgery procedures were approved by the University Animal Research Committee and meet NIH standards. Male 129S2/Sv mice (25–30 g, Charles River Laboratories, Inc. Wilmington, MA, USA) were used to induce TGI. Briefly, animals were subjected to 4% chloral hydrate intraperitoneal (i.p.) anesthesia. The body temperature was maintained at 37.0±0.5°C with heating pads, and rectal temperature was monitored throughout the surgery procedure. After midline neck incision, the bilateral CCA were carefully exposed and occluded with 6-0 suture (Surgical Specialties Co., Reading, PA, USA). The sutures were removed after 8 min for reperfusion. After wound closure, the mice were placed in an incubator set at 33.0°C and 50% humidity. They were returned to their cages after they were fully recovered from anesthesia. The sham group was preformed with the same procedure including anesthesia and all surgical procedures, except the CCA occlusion. For mitotic labeling of cell proliferation, 5-bromo-2′-deoxyuridine (BrdU, Sigma, St. Louis, MO, USA) was administered (i.p.) to animals at a dosage of 50-mg/kg body weight after reperfusion and once daily until the day of sacrifice. Animals were sacrificed by decapitation at different days after reperfusion. The brain was immediately removed and mounted in optimal cutting temperature compound (Sakura Finetek USA, Inc., Torrance, CA, USA) at —80°C for further processing.
Cerebral Blood Flow Measurement
The cerebral blood flow (CBF) was measured on both sides of the hemisphere to evaluate blood supply in the cerebral cortex before, during, and after CCA occlusion by using laser Doppler scanning imaging. The measurements and analysis were performed by using the PeriScan® system and LDPIwin 2® (Perimed AB, Stockholm, Sweden).
Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling Staining
A terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining kit (DeadEndTM Fluorometric TUNEL system, Promega, Madison, WI, USA) was used to visualize cell death in 10-μm coronal frozen sections together with NeuN. After 10-min fixing by 10% buffered formalin phosphate (Fisher Scientific, Pittsburgh, PA, USA) and pretreatment with —20°C ethanol/acetic acid (2:1) and 0.2% Triton X-100, the brain sections were incubated in an equilibration buffer as instructed by the kit. The TdT enzyme and nucleotide mix were then added at proportions specified by the kit for 75 min at room temperature. The slides were washed with the provided 2× SSC washing buffer. To identify neuronal death, slides were then incubated with the NeuN primary antibody (1:250, Chemicon, Temecula, CA, USA) overnight. After phosphate-buffered saline (PBS) washes and incubation with Cy3-conjugated antimouse immunoglobulin G (IgG; 1:1,000, Jackson ImmunoResearch, West Grove, PA, USA), the slides were incubated in Hoechst 33342 (1:20,000, Molecular Probes, Carlsbad, CA, USA) for 5 min to stain the nucleus before being mounted with ProLong Antifade mounting medium (Molecular Probes, Carlsbad, CA, USA) for observation.
Coronal fresh frozen sections of 10 μm thick were sliced using a cryostat Vibratome (Ultapro 5000; St. Louis, MO, USA). After the slides were completely air-dried, slices were fixed in 10% buffered formalin phosphate for 10 min, followed by treatments in a —20°C ethanol/acetic acid (2:1) solution for 12 min, in 0.2% Triton-100 for 5 min and washed by PBS three times between each step. Slides were blocked in 1% gelatin from cold water fish (Sigma, St. Louis, MO, USA), diluted in PBS at room temperature for 1 h, and subsequently incubated with primary antibodies diluted in PBS overnight at 4°C. The primary antibodies were as follows: mouse anti-NeuN (1:250, Chemicon, Temecula, CA, USA), goat antidoublecortin (anti-DCX, 1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-neurofilament (anti-NF, 1:1,000, Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-N-methyl-d-aspartic acid (NMDA) receptor type 1 (NR1, 1:100, Affinity BioRe-agents, Golden, CO, USA), and rabbit anti-GAD67 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After rinsing with PBS, brain sections were then treated with secondary antibodies Alexa Fluor 488 antigoat IgG (1:200, Molecular Probes, Carlsbad, CA, USA), Cy3-conjugated donkey antirabbit IgG, goat antimouse IgG (Jackson ImmunoResearch, West Grove, PA, USA), or Cy5-conjugated donkey antimouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) for 1.5 h at room temperature. Hoechst 33342 was applied to stain all the nuclei as a background staining whenever it was necessary.
To double stain DCX with BrdU, slides were postfixed and the BrdU staining procedure was performed with rat anti-BrdU (1:500, Abcam, Cambridge, UK) and Cy3-conjugated goat antirat IgG (Jackson ImmunoResearch, West Grove, PA, USA) as the primary and secondary antibodies according to our established technique [26
The brain sections were mounted and coverslipped and imaged and photographed under a florescent microscope (BX51, Olympus, Japan) and laser scanning confocal microscopy (Carl Zeiss Microimaging, Inc., Thornwood, NY, USA).
Systematic Histological Quantification
For systematic random sampling in design-based cell counting, every ninth brain sections (90 μm apart) across the entire region of interest were counted. To count BrdU-positive cells in DG, four fields per brain section were randomly chosen under ×40 magnification of a light microscope or in confocal images. This was repeated in four separate sections per brain (n=6 per group). For counting the DCX-positive cells in neocortex and BrdU-positive cells in septum, striatum, corpus callosum, and neocortex, a fluorescent confocal microscope was set to ×20 magnification, and all the immune positive cells have been counted in the particular regions (n=6 per group). Counts are expressed as the average cell number per field ± standard error (SE) of means.
Western Blot Assay of Neurogenic Factors
Expression of neurogenic proteins was examined by Western blot. Cortex tissue samples of both side hemispheres were collected, and proteins were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce Biotechnology, Rockford, IL, USA). Protein concentrations were determined by BCA assay (PIERCE, Rock ford, IL, USA). The samples were kept frozen at −80°C until assaying.
Samples of 50 μg proteins were electrophoresed on a 6–15% gradient gel in the presence of SDS–PAGE in a Hoefer Mini-Gel system (Amersham Biosciences, Piscataway, NJ, USA) and transferred in the Hoefer Transfer Tank (Amersham Biosciences, Piscataway, NJ, USA) to a PVDF membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked with buffer (Tris-buffered saline containing 0.1% Tween-20 (TBS-T), pH7.6, 7% milk) at room temperature for 2 h and incubated overnight at 4°C with rabbit polyclonal EPO (H-162), rabbit polyclonal erythropoietin receptor (EPOR; M-20), and rabbit polyclonal fibroblast growth factor 2 (FGF-2; 147; 1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-VEGF, clone JH121 (1:1,000, Upstate, Charlottesville, VA, USA), and rabbit antimouse Jun N-terminal kinase (JNK; 1:1,000, Cell Signaling, Danvers, MA, USA). Mouse β-actin antibody (Sigma, St. Louis, MO, USA) was used for protein loading control. The blots were washed in 0.5% TBS-T and incubated with alkaline phosphatase-conjugated antirabbit or antimouse IgG (Promega, Madison, WI, USA) for 2 h at room temperature. Finally, membranes were washed with TBS-T followed by three washes with TBS. Signal was detected by the addition of BCIP/NBT solution (Sigma, St. Louis, MO, USA), quantified and analyzed by the imaging software Photoshop Professional (Adobe Photoshop CS 8.0, San Jose, CA, USA).
Student's two-tailed t test was used for the comparison of two experimental groups. Multiple comparisons were done using one-way ANOVA followed by Tukey test for multiple pairwise examinations. Changes were identified as significant if p was less than 0.05. Mean values were reported together with the standard error of the mean.