Comparative effect of immature neuronal or glial cell transplantation on motor functional recovery following experimental traumatic brain injury in rats

Abstract

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.

Keywords: glia; motor functional recovery; neuron; transplantation; traumatic brain injury.

Figure 1.

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.

Figure 2.

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.

Figure 3.

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.

Figure 4.

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.

Figure 5.

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.

Figure 6.

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..

Figure 7.

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.