The present study evaluated the comparative effect of stereotaxically transplanted immature neuronal or glial cells in brain on motor functional recovery and cytokine expression after cold-induced traumatic brain injury (TBI) in adult rats. A total of 60 rats were divided into four groups (n=15/group): Sham group; TBI only group; TBI plus neuronal cells-transplanted group (NC-G); and TBI plus glial cells-transplanted group (GC-G). Cortical lesions were induced by a touching metal stamp, frozen with liquid nitrogen, to the dura mater over the motor cortex of adult rats. Neuronal and glial cells were isolated from rat embryonic and newborn cortices, respectively, and cultured in culture flasks. Rats received neurons or glia grafts (~1×106 cells) 5 days after TBI was induced. Motor functional evaluation was performed with the rotarod test prior to and following glial and neural cell grafts. Five rats from each group were sacrificed at 2, 4 and 6 weeks post-cell transplantation. Immunofluorescence staining was performed on brain section to identify the transplanted neuronal or glial cells using neural and astrocytic markers. The expression levels of cytokines, including transforming growth factor-β, glial cell-derived neurotrophic factor and vascular endothelial growth factor, which have key roles in the proliferation, differentiation and survival of neural cells, were analyzed by immunohistochemistry and western blotting. A localized cortical lesion was evoked in all injured rats, resulting in significant motor deficits. Transplanted cells successfully migrated and survived in the injured brain lesion, and the expression of neuronal and astrocyte markers were detected in the NC-G and GC-G groups, respectively. Rats in the NC-G and GC-G cell-transplanted groups exhibited significant motor functional recovery and reduced histopathologic lesions, as compared with the TBI-G rats that did not receive neural cells (P<0.05, respectively). Furthermore, GC-G treatment induced significantly improved motor functional recovery, as compared with the NC-G group (P<0.05). Increased cytokine expression levels were detected in the NC-G and GC-G groups, as compared with the TBI-G; however, no differences were found between the two groups. These data suggested that transplanted immature neural cells may promote the survival of neural cells in cortical lesion and motor functional recovery. Furthermore, transplanted glial cells may be used as an effective therapeutic tool for TBI patients with abnormalities in motor functional recovery and cytokine expression.
Traumatic brain injury (TBI) remains one of the most serious types of neurologic degeneration in humans and the deleterious effects occur during distinct primary and secondary periods (
Potential therapeutic strategies have been investigated for the treatment of patients following TBI (
These approaches have only been demonstrated to improve the neurons that remain following TBI. Therefore, successful restoration of the injured brain must be accompanied by the regeneration or transplantation of neural cells. Previous studies have suggested that genetically engineered cells, including brain tissue, embryonic stem cells, adult stem cells and neural stem cells, are capable of integrating and differentiating to restore brain function (
Conversely, another previous study found that stem cells differentiated into neural cells and increased cytokine levels in host brains (
Although various cells, including embryonic stem cells, adult stem cells and neural stem cells, have been demonstrated to improve functional recovery and protect remnant neurons in various animal models (
Therefore, to explore the therapeutic potential of immature neural cell transplantation for brain repair, the present study was undertaken to examine the comparative effect of stereotaxically transplanted neurons or glia on motor functional recovery in a rat model of TBI. Firstly, whether neurons or glia migrate into the focal injury area via brain tissue to protect the remnant neural cells and replace the lost cells was assessed. Secondly, cytokine levels were analyzed following cell transplantation to examine whether transplanted neural cells were capable of creating an environment that was conducive to functional recovery via cytokines production. Thirdly, the possible effective differences in motor functional recovery between neurons or glia transplantation were investigated.
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
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 (
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 (
The modified rotarod test is widely used to evaluate the motor coordination of rodents, and is especially sensitive in detecting cerebellar dysfunction (
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 (
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 (
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 (
No significant differences in mean rotarod scores were observed between the CON-G and the other groups prior to TBI (
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
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
GDNF expression levels decreased in a time-dependent manner; whereas TGF-β and VEGF expression levels increased with time (
The expression levels of TGF-β, VEGF and GDNF in each group, as detected by western blotting, are shown in
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.
The results of the present study demonstrated, when transplanted after the induction of TBI in rats, immature neurons or glia migrated from the injection site to the TBI lesion and significantly improved motor functional recovery, as assessed by rotarod test at 2, 4 and 6 weeks. In NC-G and GC-G, respective neuronal and glial cell transplantation significantly reduced the injury area in the ipsilateral cerebral cortex and significantly increased the expression levels of TGF-β, GDNF, and VEGF in the surrounding TBI lesion, as compared with the TBI-G. Moreover, glial cell transplantation markedly reduced the initial motor impairments induced by TBI, as compared with neuronal cell transplantation. Following cell transplantation, the rotarod scores of NC-G and GC-G increased, as compared with TBI-G. Therefore, the GC-G group resisted the decline of rotarod scores and promoted the recovery of motor function, compared with NC-G and TBI-G.
Previous studies have investigated functional recovery following stem cell transplantation in models of TBI. Undifferentiated stem cells, including bone marrow (
In the present study, first, the survival and migration ability of transplanted immature neurons or glia were evaluated, which avoided the problem of undifferentiated stem cells, in a rat model of TBI. On day 5 after TBI was established, immature neuronal and glial cells were injected into the rats far from the injury site to enhance the survival of transplanted cells. DiI, which is a lipophilic membrane stain that diffuses laterally to stain the entire cells, was used as a marker for transplanted cells. It is possible that non-transplanted cells, which graft and migrate into the brain, may pick up DiI, and stain positively. Brain sections from rats in NC-G and GC-G were double-stained with NeuN or GFAP antibodies and DiI. Transplanted cells, which were positive for NeuN or GFAP and DiI, were observed in the cerebral cortex and corpus callosum of the injection site, which is the space from the transplanted site to the injured site. Previous studies have shown that ipsilateral and contralateral transplantation of neural stem cells or human mesenchymal stem cells resulted in migration from the injection site to the injury site and significantly improved motor functions (
To investigate whether changes at the cytokine level contribute to the recovery of motor function following TBI, the effects of transplantation with neurons or glia on the expression of cytokines were assessed in rat models of TBI. The results of the present study demonstrated that transplantation of neurons or glia induced increased cytokine expression at the injury site. It has previously been demonstrated that cytokines are associated with functional recovery following brain injury (
TGF-β is a multifunctional peptide that controls proliferation, differentiation, and other functions in numerous cell types (
VEGF exerts neurotrophic effects which manifest as increased axonal outgrowth and improved cell survival in neuronal cultures (
In conclusion, the results of present study suggested that: i) Transplantation of cultured immature neurons and glia may be a potential treatment for motor functional recovery following TBI; ii) transplanted cells may have an important role in the activation of the GDNF, TGF-β, and VEGF expression; and iii) glial cell transplantation induces more beneficial effects on motor functional recovery following TBI, as compared with neuronal cell transplantation.
The present study was supported by the Youth Research Fund Project of Jilin Province (grant no. 2014-0520170JH) and the National Technology Support Project (grant no. 2015-BAI07B02).
Newly isolated cells in the culture dish differentiate into both (A) immature neurons and (B) astrocytes. Confocal microscopic images demonstrated immature cells stained with the (C) NeuN neuronal marker (green) and the (D) glial fibrillary acidic protein astrocytic marker (red) at 5 days following culture. Magnification, ×400.
Effects of neural cell transplantation on a cold-induced brain lesion at 6 weeks post-cell injection in the (A) TBI, (B) NC-G and (C) GC-G groups. (D) Quantitative analysis of the areas of the respective brain lesions demonstrated that the rats injected with (B) neuron or (C) glia cell transplants exhibited decreased lesion areas, as compared with (A) the sham TBI control. Data are expressed as the mean ± standard error of the mean. *P<0.05, as compared with the TBI-G control (n=5/group). TBI, traumatic cold brain injury; NC-G, TBI plus neuronal cells-transplanted group; GC-G, TBI plus glial cells-transplanted group; TBI-G, TBI only group.
Effects of transplantation with neurons or glia cells on (A) body weight and (B) motor function recovery with rota rod test a rat model of TBI rats. The NC-G and GC-G exhibited significant improved motor function recovery and body weight as compared with the TBI-G (*P<0.05; n=5/group). Data are presented as the mean ± standard deviation. TBI, traumatic cold brain injury; NC-G, TBI plus neuronal cells-transplanted group; GC-G, TBI plus glial cells-transplanted group; TBI-G, TBI only group; CON, control.
Number of transplanted neurons that survived up to 2, 4 and 6 weeks in different transverse planes of the (A) cerebral cortex and (B) corpus callosum and the number of transplanted glial cells that survived 2, 4 and 6 weeks in different transverse planes of the (C) cerebral cortex and (D) corpus callosum. Data are expressed as the mean ± standard error of the mean. *P<0.05 (n=5/group). W, weeks; 1/3P, the transverse plane at a 1/3 interval from the injection site to the TBI site; 2/3P, he transverse plane at a 2/3 interval from the injection site to the TBI site; TBI, traumatic cold brain injury.
Brain median sagittal section showing the fluorescent dye DiI-lableling of transplanted neural cells via corpus callosum a successful migration from the (A) injection site to the (B) traumatic cold brain injury (TBI) site. (C) Confocal microscopic images of the TBI lesion at 2, 4, and 6 weeks following cell transplantation demonstrated double-labeling of injected cells with the neuronal nuclei (NeuN) neural marker and the glial fibrillary acidic protein (GFAP) astrocytic marker. DiI-positive cells (red) in the TBI lesion were co-stained with the NeuN neuronal or GFAP astrocytic (green) markers and merged as yellow.
Effects of transplanted neurons or glia on the expression of TGF-β in brain cold injury rats. Injured rats transplanted with neurons or glia exhibited significantly increased TGF-β expression levels, as compared with the TBI-G rats. Compared to NC-G, the expression of TGF-β in the TBI lesion was significantly higher in the GC-G. No significance difference in the expression of GDNF and VEGF was detected between injured rats transplanted with neurons or glia. Notably, the confocal images showed the expression of TGF-β, GDNF and VEGF in neural cells at the injury lesion site. *P<0.05 vs. the TBI-G; #P<0.05 vs. NC-G. TBI, traumatic brain injury; NC-G, TBI plus neuronal cells-transplanted group; GC-G, TBI plus glial cells-transplanted group; TBI-G, TBI only group..
Western blot analysis was used to assess the expression levels of TGF-β, GDNF, VEGF and β-actin in the three groups. NC-G, TBI plus neuronal cells-transplanted group; GC-G, TBI plus glial cells-transplanted group; TBI-G, TBI only group; TGF. tumor growth factor.