SCF+G-CSF treatment does not improve TBI-impaired cognitive and motor functions 2 weeks after treatment
To ensure that TBI mice presented equal pathological conditions in the treated and non-treated groups before the SCF+G-CSF treatment initiation, we examined cognitive and motor functional deficits one week before treatment by Morris water maze test and Rota-Rod test, respectively. The latency to find the platform was longer in all TBI mice than in sham control mice (P < 0.01) (Additional file figure 1A). TBI mice were then randomly divided into two groups: a TBI-vehicle control group and a TBI-SCF+G-CSF-treated group. The escape latency did not show any difference between the two experimental groups (Additional file figure 1B). A similar findings was found in a 3-day pretesting Rota-Rod (Additional file figure 1C, D). These findings demonstrate the equal pathological conditions in the TBI-vehicle control group and TBI-SCF+G-CSF treatment group before the treatment initiation.
To determine the efficacy of SCF+G-CSF on cognitive and motor functional recovery, we performed Morris water maze and Rota-Rod tests 2 weeks after the treatment, respectively. In Morris water maze test, the escape latency was longer on day 4 and day 5 in TBI-vehicle control mice when compared with sham-operated mice (P < 0.05). There was no significant difference in the escape latency between TBI-vehicle controls and TBI-SCF+G-CSF-treated mice (Figure 2A). In Rota-Rod test, the latency to fall was reduced in TBI-vehicle control mice as compared to sham mice from day 2 to day 5 (P < 0.05). There was no significant difference between the TBI-vehicle group and the TBI-SCF+G-CSF group (Figure 2B). These data indicate that SCF+G-CSF treatment does not improve spatial learning and memory and motor function at 2 weeks post-treatment.
SCF+G-CSF treatment improves TBI-impaired cognitive function but not motor function 6 weeks after treatment
Next, we examined the long-term effects of SCF+G-CSF treatment on cognitive and motor functional recovery after TBI. Morris water maze and Rota-Rod tests were carried out 6 weeks after treatment.
In the Morris water maze, we found that the escape latency to platform was increased in the TBI-vehicle mice on day 1 (P < 0.05), day 4 (P < 0.05) and day 5 (P < 0.01) as compared with sham controls (Figure 2C), indicating that TBI leads to a long-term impairment in spatial learning and memory. The SCF+G-CSF-treated TBI mice, however, showed a significant reduction in escape latency when compared to the TBI-vehicle control mice on day 5 (P < 0.01, Figure 2C).These data suggest that SCF+G-CSF treatment improves TBI-impaired long-term spatial learning and memory.
In Rota-Rod test, TBI-vehicle control mice showed a decrease in latency to fall in comparison with sham controls on days 1, 2, 3 and 5 (P < 0.05). The latency to fall in TBI-SCF+G-CSF-treated mice did not differ from TBI-vehicle controls (Figure 2D). These findings suggest that SCF+G-CSF-treatment is insufficient in improving motor function after severe TBI.
SCF+G-CSF treatment does not influence the tissue loss volume after TBI
To examine whether SCF+G-CSF treatment influences the total tissue loss after TBI, we assessed the total tissue loss volume using H&E staining 20 weeks after SCF+G-CSF treatment. Our data showed that there was no significant difference in the volume of brain tissue loss between TBI-vehicle controls and TBI-SCF+G-CSF-treated mice (P > 0.05) (Additional file figure 2).
SCF+G-CSF treatment prevents TBI-induced neurodegeneration in the contralateral cortex and hippocampal CA1
In a closed-head impact TBI model, chronic traumatic encephalopathy was detected at 5.5 months post-injury [39]. In a mouse model of mild TBI, neurodegeneration was limited to the cortex 2 months after TBI, but neurodegeneration was detected in both the cortex and hippocampus at 6 months post-TBI [40]. Therefore, we chose 20 weeks after treatment (i.e. 24weeks after TBI) as the time point to evaluate neurodegeneration in the cortex, hippocampus and striatum.
To investigate the effect of SCF+G-CSF on long-term neurodegeneration after TBI, we performed Fluoro-Jade C staining 20 weeks after treatment. Fluoro-Jade C positive cells were increased in TBI-vehicle mice as compared with sham controls in both the ipsilateral (P < 0.05) and contralateral cortex (P < 0.01), in both the ipsilateral (P < 0.01) and contralateral striatum (P < 0.05), and in both the ipsilateral (P < 0.05) and contralateral CA1 (P < 0.01) (Figure 3 A-I), suggesting that the severe TBI causes a long-term neurodegeneration in both the ipsilateral and contralateral hemispheres. SCF+G-CSF treatment significantly reduced the number of Fluoro-Jade C positive cells in the contralateral cortex (P < 0.05) and CA1 (P < 0.05) as compared with TBI-vehicle control mice (Figure 3A, C, D and H). Although significant differences between the treated and non-treated TBI mice in the ipsilateral hemisphere were not seen, the Fluoro-Jade C positive cells in the ipsilateral cortex next to the TBI cavity and striatum were found no significant differences between the SCF+G-CSF-treated TBI mice and sham controls (Figure 3 E and G). These findings indicate that SCF+G-CSF treatment ameliorates severe TBI-induced long-term neurodegeneration, particularly in the contralateral cortex and hippocampal CA1.
We also analyzed neuronal loss in bilateral cortex and hippocampal CA1 by quantifying the percentage of NeuN positive cells in total of DAPI positive cells (Additional file figure 3 A-F). Surprisingly, the percentage of NeuN positive cells in the contralateral cortex and hippocampal CA1 was no difference among the sham controls, TBI control mice, and TBI mice treated with SCF+G-CSF (Additional file figure 3A, B, D, and E). The discrepancies between total degenerating neurons (Fluoro-Jade C positive) and survival neurons (NeuN positive cells) observed in the contralateral cortex and hippocampal CA1 suggest that the neuron degeneration in the contralateral hemisphere may be still in progressive process at which stage it has not reached the pathological levels to kill the neurons. By contrast to the findings in the contralateral hemisphere, NeuN positive cells in the ipsilateral cortex and hippocampal CA1 showed a similar pattern that was seen in the findings of Fluoro-Jade C positive cells in the same regions. The NeuN positive cells in the ipsilateral cortex next to the TBI cavity and the ipsilateral hippocampal CA1 were significantly reduced in the TBI-vehicle control mice as compared to the sham controls (P < 0.05, Additional file figure 3A, C, D, and F), whereas the NeuN positive cells were found no significant difference between TBI-vehicle controls and SCF+G-CSF-treated TBI mice, as well as between SCF+G-CSF-treated TBI mice and sham controls (Additional file figure 3 A, C, D, and F). These findings suggest that severe TBI may trigger a severe pathological cascade of neurodegeneration in the ipsilateral hemisphere resulting in neuron loss in the peri-TBI cavity cortex and ipsilateral hippocampal CA1.
SCF+G-CSF treatment increases apical dendritic density in the ipsilateral cortex after TBI
We then sought to determine the efficacy of SCF+G-CSF treatment in remodeling of dendritic branching after TBI. In the contralateral cortex, we did not found any differences in MAP2 positive dendrites (MAP2 positive dendritic optic density and MAP2 positive area) between sham controls, TBI-vehicle control mice, and SCF+G-CSF-treated TBI mice (P> 0.05) (Figure 4 A-E). In the ipsilateral cortex adjacent to the TBI cavity, however, TBI-vehicle mice showed significant reductions in MAP2 positive dendritic apical density in layer 1 (P < 0.01) and layer 2 (P < 0.001) as compared to sham-operated mice. This observation was further validated by analysis of MAP2 immunopositive area (TBI-vehicle controls vs. sham controls: P < 0.01 in both ipsilateral layer 1 and 2). These findings indicate that severe TBI leads to a long-term dendrites reduction in the ipsilateral cortex. SCF+G-CSF treatment, however, significantly increased MAP2 positive apical dendrites (MAP2 positive dendritic optic density and MAP2 positive area) in both layer 1 (P < 0.05) and layer 2 (P < 0.05) of the ipsilateral cortex next to the TBI cavity in comparison with TBI-vehicle controls (Figure 4F-J), suggesting that SCF+G-CSF treatment ameliorates TBI-reduced dendritic density in the peri-TBI cortex.
In the contralateral hippocampus, we did not observe any differences in MAP2 positive dendrites (MAP2 positive dendritic optic density and MAP2 positive area) among sham controls, TBI-vehicle control mice, and SCF+G-CSF-treated TBI mice in both the RAD CA1 and LM CA1 regions (P> 0.05) (Figure 5A-C, and F-H). However, in the ipsilateral hippocampus, the MAP2 positive dendritic optic density in TBI-vehicle control mice was significantly decreased in RAD CA1 (P< 0.05, Figure 5D) and LM CA1 (P< 0.01, Figure 5I) as compared to sham controls. These data were further confirmed by analysis of MAP2 positive area (TBI-vehicle controls vs. sham controls: P<0.01 in RAD CA1, Figure 5E; P< 0.05 in LM CA1, Figure 5J). These findings indicate a long-term decrease in dendritic density in the ipsilateral hippocampus by severe TBI. We did not see any increases of MAP2 positive dendritic optic density (Figure 5D and I) and MAP2 positive area(Figure 5E and J) in the ipsilateral RAD or LM CA1 after SCF+G-CSF treatment, suggesting that SCF+G-CSF does not modify the TBI-induced long-term reduction of dendrites in the ipsilateral hippocampus.
SCF+G-CSF treatment prevents TBI-induced overgrowth of axons in the ipsilateral cortex and hippocampus
To determine whether SCF+G-CSF treatment modifies axonal density after TBI, we analyzed SMI312 immunopositive axons in both the cortex and hippocampus 20 weeks after treatment (i.e. 24 weeks post-TBI).
In the contralateral cortex, the SMI312 positive axons in layer 1 were significantly increased in TBI-vehicle mice as compared to the sham controls (P < 0.05) (Figure 6A and B). TBI-vehicle mice also showed a trend toward increasing SMI312 positive axons in layer 2-3 (P = 0.081, Figure 6C) and layer 4-5 (P = 0.066, Figure 6D) in comparison with sham controls. SCF+G-CSF-treated TBI mice displayed a trend toward decreasing SMI312 positive axons in layer 1 (P = 0.087, Figure 6B) as compared to the TBI-vehicle control mice.
In the ipsilateral cortex next to the TBI cavity, TBI-vehicle mice showed significant increases in SMI312 positive axons in layer 1 (P < 0.05, Figure 6B), layer 2-3 (P < 0.05, Figure 6C), and layer 4-5 (P < 0.01, Figure 6D) when compared to sham controls, demonstrating a TBI-induced long-term overgrowth of axons in the ipsilateral cortex. SCF+G-CSF treatment, however, significantly reduced SMI312 positive axons in layer 2-3 (P < 0.01, Figure 6C), and layer 4-5 (P < 0.05, Figure 6D) as compared to TBI-vehicle control mice. A trend toward decreasing SMI312 positive axons in layer 1 (P = 0.068, Figure 6B) was also observed in SCF+G-CSF-treated TBI mice as compared to the TBI-Vehicle control mice. This observation suggests that the TBI-induced long-term overgrowth of axons in the peri-TBI cortex is inhibited by SCF+G-CSF treatment.
In the contralateral hippocampus, differences in SMI312 positive axons in the CA1 region were not seen among sham controls, TBI-vehicle control mice, and SCF+G-CSF-treated TBI mice (Figure 7A and B). In the ipsilateral hippocampus, however, TBI-vehicle mice showed a significant increase in SMI312 positive axons in CA1 (P < 0.05, Figure 7C) as compared to sham controls. SCF+G-CSF treatment significantly decreased SMI312 positive axons in the ipsilateral CA1 when compared to TBI-vehicle control mice (P < 0.05, Figure 7C). No difference was seen between TBI-SCF+G-CSF-treated mice and sham controls. These findings indicate that TBI-induced long-term overgrowth of axons in the ipsilateral hippocampus is prevented by SCF+G-CSF treatment.
To investigate axonal injury in the late subacute phase of TBI, we performed APP immunohistochemistry to identify axonal injury at 24 weeks post-TBI. Our data showed that there were no APP positive axons in the ipsilateral and contralateral corpus callosum, cortex, and CA1 (Additional file figure 4 A-C). As a control, we also performed APP immunohistochemistry in the brain sections collected from 7 days after TBI. We observed APP immunopositive staining in the ipsilateral corpus callosum and peri-TBI cortex 7 days after TBI. No APP positive staining was seen in the ipsilateral CA1, the contralateral corpus callosum, the contralateral cortex, and the contralateral CA1 at 7 days post-TBI (Additional file figure 4D). These data indicate that TBI does not induce APP expression in the injured axons in the late subacute phase of TBI. Traumatic axonal injury may occur only at the early stage of TBI but not at the late stage of TBI.