PMID: 25591773 DOI: 10.1159/000369698
Materials and Methods
Animals treatment
All the animal experiments were approved by the Institutional Animal Care and Use Committee of XuZhou Medical College. Timed-pregnant Sprague-Dawley rats were housed at 24°C on a 12-hr:12-hr light:dark cycle with free access to food and water. The PND-7 male rats (11-14g) selected from all the pups were used in the experiments. These rats were randomly assigned to control groups and ketamine groups. Ketamine was diluted in 0.9 % normal saline. PND-7 rats in treated group were administered intraperitoneally by four injections of 40 mg/kg ketamine with 1h intervals. Animals in control group received equal volume of saline at the same time points. Custommade temperature probes were used to facilitate control of temperature at 36.5 ± 1°C using computer-controlled heater/cooler plates integrated into the floor of chamber. Between each injection animals were returned to their chamber to help maintain body temperature and reduce stress.

BrdU injections
After anesthesia, neonatal rats received a single intraperitoneal injection of BrdU (5-bromo-2-deoxyuridine; Sigma, 100 mg/kg) in 0.9% NaCl solution at PND-7, 9 and 13. The animals were fixed by perfusion at 3 h after BrdU injection to observe the proliferation of matured astrocytes or at 24 h after BrdU injection to observe the proliferation and differentiation of the NSCs. The detailed experimental protocol is listed in Table 1.

Tissue preparation and double-immunofluorescence
The animals were anesthetized and then transcardially perfused after BrdU injection. The coronal sections of the brain were cut on a microtome at a thickness of 30 μm. When the SVZ was initially exposed, the five consecutive coronal sections were cut and discarded, and then the next three consecutive coronal sections were cut and selected for the double-labeled immunofluorescence of nestin/BrdU, β-tubulin III/BrdU and GFAP/BrdU, respectively. This procedure was repeated five times. The sections were incubated with 50% formamide in PBS for 2 h at 65°C and then in 2 normal hydrochloric acid incubation for 30 min at 45°C, followed by 3 washes with PBS for 10 min. The blocking of nonspecific epitopes with 10% donkey serum in PBS with 0.3% Triton-X for 2 h at RT preceded overnight incubation at 4°C, with the appropriate primary antibody listed in Table 2 in PBS with 0.3% Triton-X. After 3 washes with PBS, the sections were incubated with suitable secondary fluorescent antibodies (Alexa488-labeled donkey anti-rabbit and Alexa594-labeled donkey anti-mouse; 1:200; Invitrogen) for 2 h at room temperature. The sections were observed by a skilled pathologist blinded to this research using image stacks on a laser scanning confocal microscope (Fluoview 1000, Olympus).
Evaluation of cell apoptosis
To evaluate the effect of ketamine on apoptosis in the NSCs and astrocytes, a double-immunofluorescence detection of nestin/caspase-3 and GFAP/caspase-3 was performed. The animals were transcardially perfused with 0.9% saline followed by 4% paraformaldehyde at 12 h after the end of ketamine anesthesia. Then, the brain was removed, postfixed over-night in 4% paraformaldehyde and placed in 30% sucrose until it sunk. Coronal sections of the brain were cut on a microtome. When the SVZ was initially exposed, the coronal sections of the brain were cut consecutively at a thickness of 30 μm. The tenth section was picked up and stored in PBS for double-label immunofluorescence. The sections were blocked with 10% donkey serum in PBS with 0.3% Triton-X for 2 h at RT and then incubated overnight at 4°C with the appropriate primary antibody listed in Table 2 in PBS with 0.3% Triton-X. After being washed with PBS 3 times, the sections were incubated with the suitable secondary fluorescent antibodies for 2 h at room temperature.

Western blot analysis
The expressions of nestin, β-tubulin III and GFAP were measured using Western blot analysis. Briefly, the brain tissues from the subventricular zone (SVZ) were homogenized with lysis buffer and protease inhibitors (Beyotime, China). The lysates were centrifuged at 14000 rpm for 15 min at 4°C. Equal amounts of the proteins (25μg) were resolved on a sodium dodecyl sulfate 10% or 12% polyacrylamide gel, and the separated proteins were transferred to nitrocellulose membranes. The blots were incubated with blocking buffer for 2 h at room temperature and then incubated for 24 h with the primary antibodies against nestin (1:1000, Abcam), β-tubulin III (1:1000, Abcam), GFAP (1:1000, Millipore) and GAPDH. Then, the membranes were incubated with appropriate secondary antibodies for 1 h. The immunoreactive bands were visualized with a chemiluminescence detection system. The band intensity was quantified using Image J software.

Statistical analysis
The data are presented as the means ± SD. The statistical analysis and the graphs were completed using GraphPad Prism 5. The significant differences between the groups were analyzed with an unpaired two-tailed t-test or one-way ANOVA. P<0.05 was considered statistically significant.