New-born Sprague-Dawley rats were obtained from Laboratory Animal Centre of Xi’an Jiaotong University School of Medicine. This experiment was in accordance with the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) and the protocols were authorized by Committee on Animal Care of Xi’an Jiaotong University. The animal procedures were designed to minimize the number of animals required, and appropriate steps were taken to avoid unjustified animal procedures. This study was not involved in any vulnerable populations, for example minors, persons with disabilities or endangered animal species. Rat primary NSC cultures were prepared from the dissected cortex of Sprague-Dawley rats in 18–19 days of gestation under sterile condition. Briefly, the forebrain portion dissected and placed in ice-cold Hank’s solution (without Mg²⁺ and Ca²⁺, Gibco, Carlsbad, CA, USA). The cells were then dissociated by mechanical agitation through a fire-polished Pasteur pipette. After centrifugation, the isolated cells were resuspended in free-serum DMEM/F12 medium (Gibco, Carlsbad, CA, USA) which was supplemented with 2% B27 (Gibco, Carlsbad, CA, USA), 20 ng/ml EGF (Gibco, Carlsbad, CA, USA), 20 ng/ml bFGF (Gibco, Carlsbad, CA, USA) and 100 U/ml penicillin and phytomycin. A half of culture medium was replaced every 3 days. According to the neurosphere method developed by Reynolds and Weiss (Reynolds and Weiss, 1992), cells were incubated for 7 days to form enough neurospheres. Then the cells were passaged at a density of 2 × 10⁵ cells/ml followed by collection and dissociation. The medium was changed every 3 days. After the second passage, nestin-positive cells were enriched in neurospheres with minimal presence of β-tubulin III or glial fibrillary acidic protein (GFAP)-positive cells, which indicated the characteristic of the NSC preparation.

After passage, 5 × 10³ cells were seeded each well in 96-well plates, then Cell Counting Kit-8 (CCK-8, Beyotime Institute of Biotechnology, shanghai, China) was used to detect the cell viability by adding 10 μl CCK-8 solution per well. At the wavelength of 450 nm on a microplate reader, the Optical density (OD) value was measured 4 h later, and the value was corrected by subtracting the absorbance of control wells that did not contain cells. Data were collected from at least three independent experiments.

After passage, the cells were seeded on cover slips which processed by 100 μg/mL poly-L-lysine and incubated with the proliferation marker 5-Bromo-2′-deoxyuridine (BrdU; 10 μmol/L, Sigma-Aldrich Inc. St. Louis, MO, USA) for 4 h. Before staining with 4′,6-diamidino-2-phenylindole (DAPI) and BrdU antibody (Sigma-Aldrich Inc. St. Louis, MO, USA) cells were fixed by 4% paraformaldehyde. Immunoreactive cells were visualized by fluorescence microscopy (Olympus, BX51, Japan) and a total of 5–7 randomly selected fields were captured. The minimal number of cells for counting was 200 in each condition, and data were collected from at least three independent experiments.

After passage, the cells were seeded on cover slips which processed by 100 μg/mL poly-L-lysine and incubated with differentiating medium which contains 100 × N2 supplement, 100 × B27 supplement and 1% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) in DMEM/F12 (without b-FGF). At the end of 7 day cultures, cells were harvested for differentiation analysis. A total of 5–7 randomly selected images were taken in each well, and the number of cells expressing β-tubulin III, GFAP tested by β-tubulin III antibody (Sigma-Aldrich Inc. St. Louis, MO, USA) and GFAP antibody (Sigma-Aldrich Inc. St. Louis, MO, USA) were counted (at least 200 cells each test case), and data were collected from at least three independent experiments.

Following exposure to ketamine and/or MC, cells were washed with ice-cold D-hanks’ solution three times and then lysed with lysis buffer containing protease and phosphatase inhibitors. The cell lysates were placed on ice for 15 min and centrifuged at 14,000 rpm for 15 min at 4°C. Protein concentrations in the resulting lysates were determined with a BCA protein assay kit, bovine serum albumin (BSA) was used as a standard. Equal amount of the resulting cell lysate was resolved by sodium dodecyl sulfate-polyacrylamide gel and the separated proteins were transferred to polyvinylidene fluoride membranes. The membranes were incubated with blocking buffer for 1 h at room temperature and then incubated for 16 h at 4°C with primary antibodies (Cell Signal Technology Inc, Beverly, MA, USA) raised to β-tubulin III, GFAP, caspase-3, phospho-Akt, Akt, phosphor-Gsk-3β, Gsk-3β and β-actin. The signals were detected using goat anti-rabbit or anti-mouse horseradish secondary antibody followed by enhanced chemiluminescence (ECL). After exposing to X-ray films, blots from at least three independent tests were quantified as OD values according to their controls.

For statistical analysis, differences between animals were evaluated through one-way analysis of variance (ANOVA) and post hoc Duncon’s test. Data which expressed as the mean ± SEM were analyzed by SPSS for Windows version 18.0 and Prism 5. Statistical significance was set at P < 0.05.

To begin this experiment, neocortical tissues of E18-19 Sprague-Dawley rats were dissected for NSC culture. The suspended growth of neurospheres was notably observed 3 days after seeding. As we can see cells expressing Nestin (Figure 1A), a marker for NSCs, reached to 50%–60% of total cells in a neurosphere, which was consistent with the previous study. In the adherent culture system, NSCs also expressed Nestin (Figure 1A) after seeded on PLL. It is known that neurons, astrocytes and oligodendrocytes could be generated from NSCs by differentiation. To determine this feature of NSCs, cells were immunostained against β-tubulinIII, a specific maker for neuron (green) and GFAP, a specific maker for astrocytes (red; Figure 1B) after 7 days of differentiation. Overall, most of cells in this experiment were NSCs which characterized by the proliferation potential and the capability to differentiate into multiple cell types.

In this study, the injury of ketamine in cortical NSCs were first tested by CCK-8 (Figure 2A). Compared with vehicle controls, exposed to ketamine (from 50 μM/L to 200 μM/L, Sigma-Aldrich Inc. St. Louis, MO, USA) for 24 h significantly decreased cell viability, and 100 μM/L ketamine resulted in the minimal survival of 33.3% of NSCs. Pre-treatment of NSCs with MC (10, 20, 50, 100, 200 μM/L, Sigma-Aldrich Inc. St. Louis, MO, USA) for 30 min before ketamine exposure, the survival rate of NSCs was 39.5 ± 8%, 50.6 ± 6%, 92.8 ± 5% and 87.7 ± 8%, respectively (Figure 2B), indicating dose-dependent neuroprotective effect of MC on NSCs. It is worth to notice that there was no significant difference in the cell number between groups with different doses of MC. The maximal rescue occurred at a concentration of 50 μM/L MC. Therefore, NSCs were co-treated with 100 μM/L ketamine and 50 μM/L MC for the further study.

To evaluate the neuroprotection of MC persistently, the cell viability at 0 h, 6 h, 12 h, 24 h and 48 h were tested and we found that 100 μM/L ketamine caused a time-dependent viability decrease of NSCs but 50 μM/L MC exhibited protective effect lasting from 6 h to 48 h after ketamine exposure, besides the viability of NSCs in the control group did not change at the above time points (Figure 3A). To establish the temporal profile of MC’s effect, NSCs were exposed to 50 μM/L MC for 0.5 h, 1 h, 2 h before ketamine was added. In order to examine the acute effect of MC, it was also added to NSC culture at 0 h, 0.5 h, 1 h, 2 h after ketamine exposure. Cell viability was examined 24 h after ketamine insult. The result indicated that MC induced a time-dependent viability increase in NSCs, but considering the experimental operability and clinical practical, NSCs were pretreated with MC 30 min before ketamine exposure for the further study. It is worth noting that even when added at the same time with ketamine, NSC viability increased gently but significantly by MC treatment (Figure 3B). However, if adding at 30 min after ketamine exposure, MC did not display any protective effect against NSCs exposed to ketamine.

To investigate whether the protective effect of MC had connection with PI3K signaling pathway, LY294002 was used 120 min before ketamine exposure. The survival rate of NSCs was 92.7 ± 7% after preconditioned with MC. However, with LY294002 (20 μM/L), NSC viability was approximately 57.2 ± 6%, indicating that MC lost its protection in the presence of LY294002 (Figure 3C).

NSC proliferation was determined by BrdU incorporation staining, as an analog of thymidine, BrdU can be incorporated into the newly synthesized DNA of replicating cells during the S phase of the cell cycle. After ketamine exposure (100 μM/L; 24 h), BrdU positive cells were remarkably decreased compared to the control. On the contrary, 50 μM/L MC barely restored the proliferation rate to normal level. However, in the group pretreated with LY294002 (20 μM/L), the proliferation rate of NSCs was only 62.2 ± 7% of control group, indicating that PI3K pathway maybe involved in the protective effect induced by MC (Figures 4A,B).

Both immunostaining (Figures 5A,B) and western blot (Figures 5C,D) demonstrated that the cultured cells can differentiate into different type of cells, including astrocytes and neurons. Furthermore, it was shown that after ketamine treatment, the percentage of neuron was decreased, and MC increased the differentiation of NSCs towards neuron (Figures 5B,D). On the contrary, MC preconditioning strongly inhibited NSC differentiation towards astrocytes which was increased by ketamine exposure (Figures 5B,D). The effect of MC on NSC differentiation was reversed by LY294002, indicating that PI3K pathway maybe involved in the protective effect induced by MC (Figure 5).

Caspase-3 is often associated with apoptosis. In this study, experiments were carried out by western blot analysis to detect the difference of caspase-3 expression between three groups. Ketamine exposure produced an increase in the expression of caspase-3 compared with the control. Pretreatment with 50 μM/L MC decreased the caspase-3 expression after ketamine exposure. However, co-treatment with 20 μM/L LY294002, reversed the protective effect of MC (Figures 6A,B).

As number of previous researches indicated that the neuroprotection of MC was associated with the phosphorylation of Akt and considering that LY294002 almost completely abolished the protection of MC to NSC injury induced by ketamine, we examined the expression level of Akt/PKB, a downstream effector of PI3K which had been demonstrated to promote cell survival. In this study NSCs exposed to ketamine for 6 h lead to decreased p-Akt levels significantly, which last for 48 h in a time-dependent manner. Pretreatment with MC reversed the effect of ketamine in p-Akt levels in NSCs. Moreover, 20 μM/L LY294002 weakened the protective effect of MC for at least 48 h. There was no significant difference in total amount of Akt in our observation (Figures 7A,B).

It is known that decreased p-Akt induces lower phosphorylation of Gsk-3β to p-Gsk-3β. Therefore, the level of phosphorylated Gsk-3β was analyzed at 0 h, 6 h, 12 h, 24 h and 48 h followed by ketamine treatment. In this study NSCs exposed to ketamine for 6 h leads to decreased p-Gsk-3β level significantly, which lasts for 48 h in a time-dependent manner. Pretreament with MC reversed the effect of ketamine on p-Gsk-3β levels in NSCs. Moreover, 20 μM/L LY294002 weakened the protective effect of MC for at least 48 h. No alteration in the total amount of Gsk-3β was observed (Figures 7C,D). Our results indicated that Gsk-3β, as the crucial substrate of Akt, was involved in the protective effect of MC against ketamine insult.