A total of 252 healthy adult C57BL/6 (B6) mice (126 male and 126 female; age, 10–12 weeks), weighing 18–28 g (Vital River Laboratories, Beijing, China) were housed in a temperature- and humidity-controlled room (temperature at 21±2°C and relative humidity at 50±5%) and maintained on a 12 h light/dark schedule with free access to food and water. All experimental procedures were approved by the Animal Care and Use Committee of Third Military Medical University (Chongqing, China) and were in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

B6 NSCs were isolated from the forebrain region of 13.5-day-old murine embryos and collected by several cycles of 0.05% trypsin-EDTA (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) digestion. Cells were plated at a density of 10⁵-10⁶ cells/ml and cultured in neural stem cell culture medium (NSCcm) which was composed of DMEM/F12 (1:1) supplemented with 2% B27, 20 ng/ml basic fibroblast growth factor (bFGF) and 20 ng/ml epidermal growth factor (EGF) (all Invitrogen; Thermo Fisher Scientific, Inc.). For identification, NSCs were digested with Accutase (Invitrogen; Thermo Fisher Scientific, Inc.) and seeded on glass coverslips coated with poly-l-lysine (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) in a 24-well plate (1×10⁵ cells per well) in NSCcm for 2–3 days. The self-renewal potential of NSCs was determined by morphological assessment and immunofluorescence staining with the NSC-specific markers Nestin and Sox2. For transplantation, NSCs were digested with accutase and washed with PBS. Cells were counted by trypan blue (Sigma-Aldrich; Merck Millipore) exclusion in a haemocytometer and the density of single cell suspension was adjusted accordingly. Cells were maintained on ice.

Mice were anesthetized with 3.6% chloral hydrate (10 ml/kg; intraperitoneal injection; Sigma-Aldrich; Merck Millipore) and mounted using stereotaxic apparatus (Stoelting, Wood Dale, IL, USA). After shaving and cleaning the skin, a midline scalp incision was made using an 11 Swann scalpel and the parietal bone was exposed.

For the drilling method, an appropriate puncture point was selected on the left hemisphere, and the skull was penetrated by drilling a hole with a sterile drill (Slite P-500-3A; Ningbo Rising Trading Co., Ningbo, China) using a steel screw (diameter, 1 mm) at the speed of 12,000 rpm. For skull penetration with a syringe needle, an appropriate puncture point was selected on the right hemisphere, and the skull was penetrated with a sterile 25-µl 22S Hamilton syringe. The syringe was withdrawn once through the skull.

Mice were randomly assigned to three groups (n=84/group): Group A, receiving NSCs in the left hemisphere and the same volume of PBS in the right hemisphere; group B, receiving NSCs in the right hemisphere and PBS in the left hemisphere; and group C, receiving the equal amount of NSCs in the two hemispheres. Cell suspension or PBS was injected into the motor cortex (4.5 mm anterior to the lambda suture, 1.0 mm lateral to the middle line and 2.5 mm under the dura) using different sterile 25-µl 22S Hamilton syringes. Each site received 5 µl of cell suspension containing 5×10⁵ cells or PBS at a speed of 0.5 µl/min. Following injection for ~5 min, the syringe was slowly withdrawn. At 1, 2, 3, 7, 14, 21 and 28 days after transplantation, mice were sacrificed using a lethal dose of 10 ml/kg chloral hydrate and their brain tissues were harvested for morphological and molecular-biological analysis.

Brain tissues were postfixed in 4% paraformaldehyde in 0.1 M PBS (pH 7.4) at 4°C overnight. Tissues were sectioned (8 µm) on a cryostat (Leica CM1950; Leica Microsystems, Inc., Buffalo Grove, IL, USA) and mounted to an adhesion microscope slide. Pathological features were assessed using hematoxylin and eosin staining and viewed under a light microscope (DM2000; Leica Microsystems, Inc.) at ×200 magnification.

Cultured cells and brain slices were blocked using 10% bovine serum albumin and 0.3% Triton X-100 for 1 h, and subsequently incubated overnight at 4°C with primary antibodies (Table I). Following washing in PBS, cells and slices were incubated for 1–2 h at room temperature with secondary antibodies (Table I). Following several washes, the nuclei were stained using DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL, USA) and coverslips were mounted onto glass slides and analyzed using a fluorescent microscope (DM3000) or a confocal laser scanning microscope (TCS SP5 II; both Leica Microsystems, Inc.). The number of positive cells was manually counted at ×200 magnification directly under the microscope and adjusted using image analysis software (Image Pro Plus 5.0; Media Cybernetics, Inc., Rockville, MD, USA). The ratio of positive cells was calculated as follows: Ratio of positive cells=(number of positive cells/total number of cells) × 100%.

Brain tissues were respectively isolated from the two hemispheres and weighed. Protein was extracted from these brain tissues using radioimmunoprecipitation assay buffer (Sigma-Aldrich; Merck Millipore) supplemented with protease and phosphatase inhibitors (Fermentas; Thermo Fisher Scientific, Inc.). Protein concentration was determined using BCA assay (Thermo Fisher Scientific, Inc.). Mouse brain-derived neurotrophic factor (BDNF) glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), interleukin-6 (IL-6), interleukin (IL)-10 and tumor necrosis factor alpha (TNF-α) were detected using ELISA kits. BDNF ELISA kit was purchased from Promega Corp., (Madison, WI, USA). GDNF ELISA kit was purchased from Santa Cruz Biotechnology Inc., (Dallas, TX, USA). NT-3 ELISA kit was purchased from Abcam, (Cambridge, MA, USA) and IL-6, IL-10 and TNF-α ELISA kits were purchased from Sigma-Aldrich (Merck Millipore). ELISA was carried out in duplicate, according to the manufacturers' protocols.

The SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA) package was used for statistical analysis. Data were presented as mean ± standard deviation. Student's t-test and one-way analysis of variance were used to determine statistical significance. P<0.05 was considered to indicate a statistically significant difference.

B6 NSCs were isolated from the forebrain region of 13.5-day-old murine embryos and had self-renewal potential. These cells formed neurospheres that grew in culture medium in a state of suspension. The results of immunofluorescence staining indicated that these cells were strongly positive for Nestin and Sox2 (data not shown).

Macroscopic inspection showed obvious injuries and hemorrhage in the left hemisphere during the early stage of post-implantation for all the groups (data not shown). Wounds observed were irregular, unstable and varied within the drilling method group; whereas in the right hemisphere, wounds were small and exhibited a similar appearance to one another following syringe needle penetration. Histological examination of all groups indicated that a small amount of bleeding was observed in the first two days in the right hemisphere, which underwent syringe penetration. In contrast, the structure of the left hemisphere, which underwent drill penetration, was broken and exhibited extensive necrosis, infiltrated immune cells and hemorrhage. After one week, scar tissue formation was observed in all groups, and only a small number of neural cells were detected in the left hemisphere, whereas the contralateral cells were well-arranged. After 28 days, neural cells were rarely observed in the injured area, which was filled with massive scar tissue in the left hemisphere. In addition, brain injuries in the left hemisphere of mice receiving PBS treatment were notably worse than those receiving NSC transplantation.

To measure the extent of inflammation secondary to brain injury after implantation, the distribution of infiltrated immune cells, including microglia/macrophages [cluster of differentiation (CD)11b], T cells (CD3) and B cells (CD19) was observed. The results of group C in the left and right hemispheres are presented in Fig. 1A and B, respectively. The number of immune cells was elevated in the left hemisphere; however, these cells were rarely detectable in the right hemisphere in all groups. As demonstrated in Fig. 1C-E, in group A, the three types of immune cells (CD11b, CD3 and CD19, respectively) increased rapidly in the first three days and remained at a high level for 2–3 weeks. In group B, the difference between the two hemispheres was the most obvious and the number of immune cells in the left hemisphere was the highest of the three groups. Only a small number of infiltrated immune cells were detected in group C, the difference between the two hemispheres in this group was statistically significant (P<0.05). In addition, the number of immune cells in the part of the hemisphere receiving NSC transplantation was lower than that in the same side receiving PBS treatment (P<0.05).

Immune cells were predominantly distributed around the broken structure of the skull and were positively correlated with the degree of injury. An increase in the number of immune cells observed suggested that inflammation secondary to skull penetration using a drill was significantly more severe when compared with skull penetration using a syringe needle (P<0.05). The number of microglia and macrophages increased in the first week but the peak remained only for a short time (Fig. 1C). T cells were noticeable at the early stage following brain injury; however, the level of T cells did not change significantly throughout the study period (Fig. 1D). Compared with the other immune cells, the number of B cells was lower and the peak appeared later in three weeks (P<0.001; Fig. 1E). In addition, the number of immune cells varied within the left hemisphere treatment group among all the groups (groups A-C).

To investigate the effects of transplantation approaches on hosts and grafts, the levels of neurotrophic and immunomodulatory factors in the cerebral cortex were detected (Fig. 2). ELISA results revealed that BDNF, GDNF and NT-3 concentrations in the right hemisphere of group B and C increased significantly (P<0.05; Fig. 2A-C, respectively), whereas there was no significant difference in BDNF, GDNF and NT-3 concentrations between the two hemispheres in group A. Levels of neurotrophic factors in the NSC transplantation hemispheres were higher than those in the PBS treatment side (P<0.05; Fig. 2A-C).

Furthermore, immunomodulatory factors including pro- and anti-inflammatory cytokines increased significantly in the left hemisphere in response to the infiltrated immune cells among all the groups (P<0.05). The levels of pro-inflammatory cytokines (IL-6 and TNF-α) in the PBS injected hemisphere were significantly higher than those in the NSC transplantation side (P<0.05; Fig. 2D and E, respectively). However, the levels of anti-inflammatory cytokine IL-10 in the PBS injected hemisphere were significantly lower than that in the NSC transplantation side (P<0.05; Fig. 2F).