A total of 60 male Sprague-Dawley rats, weighing ~220 g and aged 7 weeks±2 days, were purchased from the Experimental Animal Center of the College of Animal Sciences at Jilin University (Changchun, China) and were used in the present study. Rats were maintained at 22°C (humidity, 60%) with a 12-h light/dark cycle and ad libitum access to food and tap water. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Jilin University.

Rats were divided into four groups (n=15/group): i) Sham (CON); ii) TBI plus neuronal cells-transplanted group (NC-G), rats were transplanted neuronal cells 5 days after TBI; iii) TBI plus glial cells-transplanted group (GC-G), rats were transplanted glial cells 5 days after TBI; iv) TBI only group (TBI-G), rats received TBI only. Five rats from each group were sacrificed at 2, 4 and 6 weeks after the graft via an overdose of sodium pentobarbital (30 mg/kg; Abbott Laboratories, Chicago, IL, USA).

Cortical neuron cultures were harvested from the brains of 16-day-old rat embryos according to a modified procedure outlined by Freshney in 1987 (23). Briefly, cerebral hemispheres were isolated and placed into Ca²⁺/ Mg²⁺-free Hank's balanced salt solution (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Brain tissue was dissociated in 0.025% trypsin for 10 min at 37°C and the proteolytic reaction was subsequently terminated by adding the same quantity of Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum (FBS; both Gibco; Thermo Fisher Scientific, Inc.). Following centrifugation at 157 × g for 15 min at 4°C, the pellet containing the dissociated neuronal cells was resuspended in neurobasal media containing 400X L-glutamine (200 Mm), 50X B27, 100X penicillin and streptomycin antibiotics (all Gibco; Thermo Fisher Scientific, Inc.) and 200X glutamate (5 Mm; Sigma-Aldrich RBI, Natick, MA, USA). The concentration of the cells was adjusted to 2×10⁶ cells/ml and the viability of the cells was 85%, as determined by the trypan blue dye exclusion method (24). Cells were seeded onto a poly-D-lysine (50 µg/ml; Sigma-Aldrich, St. Louis, MO USA) and lamine (1 µg/ml; Gibco; Thermo Fisher Scientific, Inc.)-coated 6-well plastic plate at a concentration of 1×10⁶ cells/well and maintained at 37°C in an atmosphere containing 95% air and 5% CO₂.

Mixed glial cell cultures were prepared from dissociated cerebral cortices of newborn rats. Dissociation of the cerebral hemispheres and cell culture were performed as described above for the cortical neuronal cells, although the components of the culture media were altered to DMEM supplemented with 10% FBS and 1% antibiotic solution (100X; Gibco; Thermo Fisher Scientific, Inc.).

Cultured neural cells were labeled with DiI fluorescent dye (1,1-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine, perchlorate) prior to injection to ensure the cells appeared red. A total of 45 rats were randomly selected, anesthetized with 8% chloral hydrate (400 mg/kg) by intraperitoneal injection and placed in a stereotaxic frame. Microinjection coordinates (1 mm posterior, 1.5 mm right lateral, 2 mm ventral to the bregma) were selected according to a rat brain atlas and microinjection was performed using a 20 µl Hamilton syringe with a 22-gauge needle. Five days after TBI, rats received a microinjection of 10 µl cell suspension (~1×10⁶ cells). The cell suspension was separated into 5 parts and injected slowly over 25 min with 5 min intervals for each 2 µl cell suspension.

In the present study, a well-characterized traumatic injury model was used, as previously described (27). This injury model was selected as it is readily reproducible and allows for the comparison of affected (ipsi-) and non-affected (contralateral) hemispheres. Following intraperitoneal injection with 10% chloral hydrate (3.5 ml/kg) anesthesia, the head was fixed in a stereotaxic frame. Subsequently, the scalp was longitudinally incised and the indiction was made on the right motor cortex (4 mm anterior from the lambdoid suture; 2 mm lateral from the midline). A probe (diameter, 4-mm) was pre-cooled with liquid nitrogen and used to touch the surface of the skull for 20 sec. Subsequently, the skin was sutured using 3.0 nylon in a running whipstitch and the rats were observed until they awoke.

The modified rotarod test is widely used to evaluate the motor coordination of rodents, and is especially sensitive in detecting cerebellar dysfunction (26). All the rats underwent conditioning of the rotarod test for three trials a day for 5 days prior to TBI. Motor behavior was analyzed daily for three trials on days 1, 5, 7, 14, 28 and 42 following TBI by a blinded observer using rotarod apparatus (Med Associates, Inc, St. Albans, VT, USA). Rats were placed on a rotating rod which was set to slowly accelerate from 4–40 rpm over 5 min. The rotarod test requires the rat to walk and maintain balance as the revolving rod accelerates. The trial was terminated when the rat fell off the apparatus and tripped a plate which recorded the time.

At 2, 4 and 6 weeks following neural cell injection, rats were anesthetized with intraperitoneal injection of 10% chloral hydrate (3.5 ml/kg) and intracardially perfused with 150 ml normal saline and 300 ml ice-cold 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4). The brains of the rats were removed and post-fixed in the same fixative overnight at 4°C. Brain images were captured using a digital camera. Necrotic areas were measured using morphometry software (Axiovision 3.0.6 SP4; Carl Zeiss AG, Oberkochen, Germany). For light microscopy studies, tissues were embedded in paraffin blocks.

For immunofluorescence imaging, 5 µm-thick paraffin-embedded brain sections were cut, deparaffinized and rinsed in PBS. Sections were incubated in PBS supplemented with 3% bovine serum albumin (BSA), 10% normal calf serum and 1% Triton X-100 for 1 h at room temperature to block nonspecific binding. Subsequently, the sections were incubated overnight at 4°C with mouse anti-neuronal nuclei (NeuN; 1:500; ab18956; Abcam, Cambridge, UK) and rabbit anti-glial fibrillary acidic protein (GFAP; 1:1,000; MA191029; Chemicon International, Inc., Temecula, CA, USA) monoclonal antibodies in PBS containing 1% Triton X-100. Following washing in PBS for 30 min (6 times for 5 min), the sections were incubated with fluorescein isothiocyanate-conjugated, affinity-purified anti-mouse IgG and anti-rabbit IgG (both 1:200; both Jackson Immuno-Research Laboratories, West Grove, PA, USA) for 2 h at room temperature. Following washing in PBS for 30 min, the sections were mounted with mounting medium and examined under a fluorescence microscope (Olympus BX51; Olympus Corporation, Tokyo, Japan).

Sections (5 µm) were cut, mounted on positively-charged slides, air-dried in an incubator at 40°C overnight and deparaffinized in xylene. Following rehydration with a graded alcohol series, the slides were incubated with 1% hydrogen peroxide diluted in methanol for 15 min to block endogenous peroxidase activity, and were subsequently rehydrated in distilled water and PBS. Following this, the slides were incubated with a blocking solution (PBS supplemented with 3% BSA and 10% normal calf serum) for 60 min at room temperature. This solution was removed from the slides using filter paper and the samples were incubated with the respective primary antibodies overnight at 4°C. The following primary antibodies were used: Mouse anti-glial cell-derived neurotrophic factor (GDNF; ab18956), rabbit anti-transforming growth factor (TGF)-β (both 1:500; ab66043) and mouse anti-vascular endothelial growth factor (VEGF; 1:400; ab46154; all Abcam). Following removal of the unbound antibodies via several rinses with PBS followed by PBS containing 0.1% Triton X-100, the respective antibodies were detected using the avidin-biotin-peroxidase complex method (orb90540) and visualized using 3,3′-diaminobenzidine. Slides were lightly counterstained with hematoxylin.

Double-labeled sections were observed with an Olympus BH2 light microscope or an Olympus FV-1000 confocal laser-scanning microscope. The five darkest points of the images obtained by light microscopy were analyzed via densitometry using Multi Gauge 3.0 software (Fujifilm Life Science, Minato, Tokyo, Japan).

The cortex of each rat at the injury site was harvested on ice. Following washing with 0.9% sodium chloride, the clean tissue was preserved in liquid nitrogen until use. A protein preparation kit was used to exact the protein, and the concentration was measured by the Bradford method. Protein samples (20 µg) from cortex of each rat at injury site were separated by 12% gel electrophoresis and transferred on to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked in Tris-buffered saline with Tween 20 (TBS-T; pH 7.4) supplemented with 3% skimmed milk, and subsequently incubated overnight at 4°C with anti-TGF-β, anti-GDNF and anti-VEGF primary antibodies (1:500). Following washing, the membranes were incubated with the following secondary antibodies: Anti-mouse IgG (CABT-ZC1010) for GDNF and VEGF and anti-rabbit IgG (CABT-ZC1022) for TGF-β (1:2,000; Jackson Immuno-Research Laboratories) for 2 h at room temperature. All antibodies were diluted in TBS-T supplemented with 3% skimmed milk. The membranes were developed using enhanced chemiluminescent reagent and subjected to autoluminography for 2 min. Membranes were exposed on X-ray film (Eastman Kodak Company, Rochester, NY, USA). Blots were subsequently striped and re-blotted with mouse anti-β-actin primary antibody (1:5,000; ab54724; Abcam), followed by incubation with anti-mouse IgG secondary antibody (1:2,000; PA128555; Thermo Fisher Scientific, Inc.).

Data were analyzed using SAS 8.1 (SAS Institute Inc., Cary, NC, USA) with analysis of variance. P<0.05 was considered to indicate a statistically significant difference. Data are presented as the mean ± standard deviation.

The isolated cells were successfully induced to differentiate into immature neurons and glia, respectively (Figs. 1A and B). Neurons and glia were stained with NeuN neuronal marker and GFAP astrocyte marker, respectively (Figs. 1C and D).

Establishment of the rat model of TBI resulted in a wedge-like cold brain lesion which affected the motor cortex. The dorsal view of the brain 6 weeks post-TBI demonstrated that there was a marked difference in the injury area between the TBI-G and the NC-G and GC-G (Figs. 2A-C). The NC-G and GC-G exhibited decreased injury areas, as compared with the TBI only control group, TBI-G. Quantitative analysis of the lesion area 6 weeks post-TBI demonstrated that transplantation with neuronal and glial cells following TBI significantly reduced the injury area in the cerebral cortex (P<0.05; Fig. 1D).

As compared with the rats in NC-G and GC-G, the mean body weight of the rats in the TBI-G was lowest from the day of TBI administration to 6 weeks post-TBI, and a notable decrease was observed at 2 weeks post-TBI (Fig. 3A). The body weight of rats in the CON-G remained significantly higher than other groups across all the time points (P<0.05). The NC-G and GC-G demonstrated significantly higher body weight increases, as compared with the TBI-G at 2 weeks post-TBI (P<0.05). Rats in the GC-G exhibited significantly increased body weight at 4 and 6 weeks post-TBI, as compared with the NC-G.

No significant differences in mean rotarod scores were observed between the CON-G and the other groups prior to TBI (Fig. 3B). Following TBI, TBI-G, NC-G and GC-G demonstrated a significant decline in rotarod performance, as compared with their pre-TBI scores. Rotarod scores of rats in NC-G and GC-G were significantly higher than TBI-G at 2, 4, 6 weeks post-cell transplantation (P<0.05). Furthermore, the GC-G demonstrated a reduced decline in rotarod scores, as compared with NC-G and TBI-G, suggesting glial cell transplantation promoted the recovery of motor function. No significant changes in rotarod performance were detected for rats in the CON-G at any time.

Migration ability of transplanted-neurons or transplanted-glia were observed vai the longitudinal view of the brain at 2, 4 and 6 weeks post-cell transplantation. As shown in Fig. 4, transplanted cells migrated via the corpus callosum from the injection site to the TBI lesion at 2 weeks. The number of transplanted-neurons and transplanted-glia at injection site decreased, whereas the number of these cells increased with time at the TBI site. The number changes of neurons and gila with labeling Dil in cortex and corpus callosum were similar.

DiI pre-labeled neuronal or glia were observed at the injection site and surrounding areas of TBI injury 2, 4 and 6 weeks post-transplantation. In NC-G, DiI-positive cells exhibited immunoreactivity for the NeuN neuronal marker and substantial co-expression, as shown in Fig. 5; however these cells did not exhibit immunoreactivity for GFAP. In GC-G, the pre-labeled glia were immunoreactive for the GFAP astrocytic marker but did not exhibit immunoreactivity for NeuN. These results demonstrated that the transplanted neurons and glia, although small in population, successfully migrated from the transplant site to the TBI lesion and differentiated into neural cells in vivo following TBI.

GDNF expression levels decreased in a time-dependent manner; whereas TGF-β and VEGF expression levels increased with time (Fig. 6). Quantitative analysis demonstrated that TBI-G, NC-G and GC-G exhibited variable TGF-β (P<0.05), GDNF (P>0.05) and VEGF (P>0.05) expression levels at 2, 4 and 6 weeks post-transplantation. NC-G and GC-G exhibited significantly increased secretion of the three cytokines, as compared with TBI-G at the same time points. Moreover, GC-G treatment significantly increased the expression of TGF-β, GDNF and VEGF, as compared with NC-G.

The expression levels of TGF-β, VEGF and GDNF in each group, as detected by western blotting, are shown in Fig. 7. The expression levels of CON-G. TGF-β, VEGF and GDNF were increased in NC-G and GC-G, as compared with the CON-G. TGF-β, VEGF and GDNF expression levels were increased in GC-G, as compared with NC-G.

Confocal microscopy demonstrated the expression of TGF-β, VEGF and GDNF in neural cells at the injury site (data not shown). TGF-β, VEGF and GDNF expression was observed in transplanted-neurons and transplanted-glia. These results indicated that transplanted-neurons and glia may secrete TGF-β, VEGF and GDNF.