| Objective The Raf/Erkl/2/Merk signaling pathway controls many cellular responses such as cell proliferation, migration, differentiation and death. In the nervous system, emerging evidence also points to a death-promoting role for Erk1/2in both in vitro and in vivo models of neuronal death. To further investigate how Raf/Erkl/2/Merk up-regulation may lead to the development of spinal cord injury, we developed a cellular model of Raf/Erk up-regulation by over-expressing c-Raf in cultured SMNs (SMNs) and astrocytes (ASTs). In order to explore the role of Raf/Erkl/2/Merk signal pathway on the nerve regeneration in animal models of spinal cord injury, We delvelped the animal model of spinal cord injury, by using Raf/Erkl/2/Merk signal pathway inhibitors, to observe in mice spinal cord injury axonal regeneration and functional recovery after SCI.Methods Spinal cord motor neurons (SMNs) and astrocyte (ASTs) were prepared from C57BL/6J mouse pups (Dl). SMNs and ASTs were infected with Ad-Raf-1or Ad-GFP adenovirus alone. Cell adhesion assay and cell migration assay were investigated, DiI labeling was imployed to to exam the effect of up-regulation of Raf/Erkl/2/Merk signaling on the dentrific formation of spinal neurons. We used the TO-PRO-3staining to exam the apoptotic effect of c-Raf on SMNs. The effect on the synapse formation of neurons was measured by using Immunofluorescence, western-blot and patch clamp. Then, SCI model in C57BL/6J mice (8weeks’ old) was developed at T10with NYU Impactor-Ⅱ machine with10g×5mm weight drop.60SCI mice were classified into3groups randomly and evenly:None surgery group(DMSO), Surgery group(DMSO), Surgery group(U0126). BMS score evaluation and Rotard test were carried out at before and after injury.1,2,3,4weeks after operation,2animals of each group were sacrificed, and injured spinal cord tissue was used for Immunoflurecense, Western-Bolt, Real-time PCR analysis and patch clamp.12weeks later,8mice in each group were chosen out to carry out10%BDA anterograde tracing mark. Then,2weeks later, the animals were sacrificed, the spinal cord tissue with injured lesion was taken out and fast frozen sections(5μm) was made followed by BDA developing. The pictures were processed by microimage analysis software (ImageJ5.0).Results We Successfully separated and purified spinal motor neurons and spinal cord astrocytes cells from mice pups.The intracellular Raf/Erk1/2/Merk signal level was upregulated. The increase of the Raf/Erk1/2/Merk signal level promoted the migration ability of spinal motor nerve cells and glial cells, but impeded the project formation of the spinal cord motor neurons, inhibited the maturation of spinal cord motor neurons dendrits. But as the Raf/Erk1/2/Merk signal level rises, we found that astrocytes in vitro were significantly enhanced,and promoted the development and maturation of astrocytes. After co-culture of72h with astrocytes, the markers of spinal motor neurons synapse were increased and emerged the phenomenon of clusters, further promoted differentiation of axons and dendrites. The number of synapse was increased by7-fold compared with the control group (p<0.001). In animal models, U0126group has more nerve fiber regeneration through the damage zone, and spinal cord injury group and non-surgery group almost no nerve fiber regeneration through. The immunohistochemical staining showed damage voids in U0126group was significantly smaller than the other two groups (p<0.05).Conclusion These results suggest that the up-regulation of the Raf/Erk1/2/Merk signaling pathway may contribute to the pathogenesis of spinal cord injury through both its impairment of spinal cord neuron development and causing neural circuit imbalances. |
PDF Full Text Request