Focal cerebral ischemia procedure
Animal experiments were approved by the Animal Care Committee (Institute of Science and Technology, Jiangsu Province, China). All procedures were performed under the guideline published in the NIH guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, revised 1985). Every effort was made to minimize pain and discomfort.
Adult male C57BL/6 mice (n = 50) weighing 25–30 g (Model Animal Research Center of Nanjing University) were used for all experiments. Mice were anesthetized with chloral hydrate (10 %, 400 mg/kg, intraperitoneally). Stroke was induced by intraluminal middle cerebral artery occlusion (MCAO) with a monofilament according to Zea Longa . In brief, the right common carotid artery, the right external carotid artery and the internal carotid artery were exposed via a midline incision. A 8-mm long 8-0 monofilament, with its tip rounded by heating near a flame, was advanced from the external carotid artery stump into the right internal carotid artery to occlude the origin of the right middle cerebral artery. Ninety minutes after occlusion, the filament was withdrawn to allow reperfusion. The rectal temperature was maintained at 37.0–37.5°C with a heating pad throughout the surgical procedures.
Animals were randomly divided into control (n = 30) and TGF-β1 groups (n = 20). Recombinant human TGF-β1 (PeproTech Inc, USA) was dissolved in normal saline to a final concentration of 50 μg/ml. Intranasal administration was performed as described previously with some modifications [22, 23]. Two hours after MCAO, mice were re-anesthetized with chloral hydrate (10 %, 200 mg/kg, intraperitoneally) and placed on their backs. A total volume of 20 μl solution per mouse containing 1 μg TGF-β1 or saline was given as 2 μl drops into the left and right nares, alternating sides at 2 minutes intervals over a period of 20 minutes. The mouth and the opposite naris were shut during the administration. A second series of doses was given 24 hours after initiating MCAO.
BrdU (50 mg/kg, ip; Sigma, St. Louis, MO, USA) was dissolved in saline and given intraperitoneally twice daily at 8 hour intervals for three consecutive days, starting 24 hours after initiating MCAO.
Each mouse was subjected to the NSS test to evaluate neurological function before and at 1, 4, 7, 14, 21, 28 days after the onset of MCAO , and the scores were assessed by another investigator who was unaware of the experimental groups. NSS is a functional evaluation composite of motor, sensory, reflex and balance tests. The score was graded from 0 to 18 (normal score, 0; maximal deficit score, 18). Severe injury is indicated by a score of 13 to 18, moderate injury 7 to 12, and mild injury 1 to 6. In the severity scores of impairment, one point is scored for the inability to perform the task or lacking proper response for a given reflex.
Tissue preparation and infarct volume measurement
At 7, 14, 21 or 28 days after stroke, mice (n = 6 in control group and n = 4 in TGF-β1 group at each time point) were deeply anesthetized with chloral hydrate and perfused transcardially with 150 ml saline, followed by 150 ml 4 % paraformaldehyde in 0.01 M phosphate-buffered saline (PBS) at 4°C. Brains were post-fixed in 4 % paraformaldehyde for 6 hours and cryoprotected in 30 % sucrose solution overnight. Twenty micrometer coronal sections, spaced 200 μm apart, encompassing the SVZ were cut with a cryostat and stored at -80°C.
Every 40th coronal section from total brain was stained with hematoxylin. The infarct area, and the contralateral intact hemispheric area were measured by a blinded observer using the NIH Image program. The infarct volumes were calculated by multiplying the interval thickness and presented as a percentage of the intact hemisphere, as described previously .
The brains were carefully removed at 7 days after MCAO. Five 20 μm coronal sections per brain were cut on a cryostat. TUNEL staining was preformed using a Roche assay kit (In situ Cell Death Detection Kit; Roche, USA), according to the manufacturer's protocol.
Five to seven DAB-stained, 20 μm sections per animal were taken to evaluate the number of BrdU-labeled cells. For BrdU immunostaining, brain sections were treated with 50 % formamide, 280 mmol/L NaCl and 30 mmol/L sodium citrate at 65°C for 2 hours, incubated in 2 mol/L HCl at 37°C for 30 minutes, rinsed in 0.1 mol/L boric acid (PH 8.5) at room temperature for 10 minutes. After incubating in 3 % H2O2 for 30 minutes, sections were blocked in PBS containing 2 % goat serum, 0.3 % Triton X-100, and 0.1 % bovine serum albumin for 1 hour, followed by incubation with mouse monoclonal anti-BrdU antibody (1:800; Sigma) at 4°C overnight. Biotinylated goat anti-mouse secondary antibody (1:500; Jackson ImmunoResearch, West Grove, PA, USA) was applied for 2 hours at room temperature, then washed and incubated with peroxidase-conjugated streptavidin solution (1:500; Jackson ImmunoResearch, West Grove, PA, USA) for 30 minutes. Reaction product was detected with 3, 3'-diaminobenzidine- tetrahydrochloride (DAB, Sigma).
For double immunofluorescent staining, sections were pretreated for BrdU immunohistochemistry as described above. The following primary antibodies were used in this study: mouse monoclonal anti-BrdU antibody (1:800; Sigma), sheep polyclonal anti-BrdU antibody (1:800, Biodesign, Saco, ME), mouse monoclonal anti-NeuN antibody (1: 600, Chemicon, Temecula, CA, USA), guinea pig polyclonal anti-DCX antibody (1:3000, Chemicon), mouse monoclonal anti-GFAP antibody (1:800, Chemicon). The secondary antibodies were fluorescein isothiocyanate-conjugated goat anti-mouse IgG (1:100, Jackson ImmunoResearch), rhodamine-conjugated donkey anti-sheep IgG (1:100, Jackson ImmunoResearch), Cy3-conjugated goat anti-guinea pig IgG (1:100, Jackson ImmunoResearch). Sections were mounted with Vectashield Mounting Medium H-1000 (Vector Laboratories, Burlingame, CA, USA).
Sections were viewed under high power (200×) on an Olympus BX51 microscope with Nikon digital camera, and the images were visualized on a computer monitor. The numbers of TUNEL-positive cells were counted in the impaired striatum of each animal. For BrdU-staining, only the cells with BrdU clearly localized and confined to the nucleus were considered as BrdU-reactive cells. All of the BrdU-positive cells in the lateral ventricle wall (SVZ) and striatum ipsilateral to the injury were counted. Results are presented as the average number of TUNEL or BrdU-positive cells per section.
Double-labeled cells with BrdU and a phenotype-specific marker (DCX, NeuN, or GFAP) in brain sections were identified using a confocal laser-scanning microscope (Leica TCS SP2, Germany). Only the cells in which BrdU staining was strong and clearly distributed throughout the nucleus were counted. Results are expressed as percentages of BrdU-labeled cells.
Values for all animals of each group were averaged and standard errors of mean (SEM) were calculated for each endpoint. Statistical analysis was carried out by using two-way analysis of variance (ANOVA), followed by pairwise Student's t-test for modified NSS. Infarct volume and cell numbers comparisons were performed with Student's t-test. Probability values of < 0.05 were considered significant.