The Experimental Study Of The Role And Mechanism Of ERK1/2 Pathway In Diffuse Brain Injury Of Rats

Objective To investigate the feature of ERK1/2 pathway activation in traumatic diffuse brain injury in rats by determining the expression of phosphorylated extracellular signal-regulated kinase1/2(phospho-ERK1/2 ), a key kinase of ERK1/2 cascade.Methods Marmarou's free falling-body model was used to induce diffuse brain injury. 58 male Sprague-Dawley rats (320g-370g weight) were divided into two groups randomly, including sham operated group as control, diffuse brain injury group. Western blot analyses and immunochemistry were used to evaluate the expression level and regional distribution of phospho-ERK1/2 in injured brain tissue respectively. Determining the categories of phospho-ERK1/2 positive-immunostained cells by double-labeled immunofluorescent staining method (phospho-ERK1/2 and NeuN combined, phospho-ERK1/2 and GFAP combined).Results Immunoblot analyses demonstrated that phospho-ERK1/2 protein was significantly increased , and reached top as early as 5 min, lasting about 72 h after brain trauma. Immunohistochemical evidence of robust activation of ERK1/2 pathways was observed predominantly in the region of deep layer of cortex, then in hippocampus. Ependyma and choroid plexus cells were also positive immunostained. Double-labeled immunofluorescent staining method showed phospho-ERK1/2 colocalized with NeuN and GFAP respectively.Conclusions ERK1/2 pathway was over- and sustained- activated in traumatic diffuse brain injury. pERK1/2 positive immunostained cells mainly localized in the deep layer of cortex, then in hippocampus. pERK1/2 colocalized with NeuN and GFAP respectively. Objective To explore the role of U0126, a specific inhibitor of ERK1/2 pathway, in diffuse brain injury of rats after trauma, in the following aspects: neuronal injury, apoptosis and reactive astrogliosis.Methods Sprague-Dawley rats(320—370g weight) were randomly divided into four groups: sham operated (sham group), traumatic diffuse brain injury(DBI group), DBI and treatment with U0126(U0126 group), vehicle treated as control (DMSO group).Rats were subjected to diffuse brain injury by Marmarou's free falling-body method. DBI and U0126 groups were further divided into seven subgroups at different time points after trauma (5 min, 30 min, 3 h, 12 h, 24 h, 72 h, 7 d). U0126 (including 0.05mg/kg and 0.1mg/kg dose) was administered intravenously through caudal vein 30 min before trauma. Western blot analyses and immunochemistry were used to evaluate the expression level and regional distribution of phosphorylated ERK1/2 in brain tissue after trauma. The expression of NSE, Caspase-3 and GFAP were evaluated by immunochemistry immunostaining. Transmission electron microscope was used to observe the microstructure of injured neurons. Results Western blots analyses illustrated phophorylated ERK1/2 was significantly increased, peaking at 5 minute, and sustained for 72 hours after trauma. Treatment with U0126 effectively prevented the activation of ERK1/2 dose-dependently (P < 0.01 or P < 0.05) . Immunochemistry staining results showed the expression of NSE increased at 30 min after trauma, then decreased. Treatment with U0126 before trauma can stabilize the expression level of NSE between 12 h and 24 h intervals. The expression of Caspase-3 was inhibited by U0126 at 24 h and 72 h following trauma (P < 0.05) . The expression of GFAP increased from 12 h time point, and reach a top at 72 h. U0126 reduced the level of GFAP significantly from 12 h to 72 h (P < 0.05) . Finally, the protective role of U0126 on neuron injury was observed by transmission electron microscope.Conclusions ERK1/2 pathway was over- and sustained- activated in diffuse brain injury after trauma. Inhibition of this pathway by U0126 can protect neuron against secondary injury, attenuate reactive astrogliosis and the the expression of Caspase-3. Objective To study the effect of U0126, a selective inhibitor of extracellular signal-regulated kinase1/2 (ERK1/2) pathway, on the expression of c-fos gene in traumatic diffuse brain injury of rats.Methods Marmarou's free falling-body methods was used to induce brain injury. Sprague-Dawley rats(320-370g weight) were randomly divided into four groups: sham operated (sham group), traumatic diffuse brain injury(DBI group), treatment with U0126 before trauma(U0126 group), vehicle treated as control (DMSO group). DBI and U0126 groups were further divided into seven subgroups at different time point after trauma(5 min, 30 min, 3 h, 12 h, 24 h, 72 h, 7 d). U0126 (including 0.05mg/kg and 0.1mg/kg dose) was administered intravenously through caudal vein 30 min before trauma. The expression of phosphorylated ERK1/2 and c-fosmRNA was detected by Western blot and RT-PCR methods respectively. Positive immunostained cells of phosphorylated ERK1/2 and c-fos in damaged brain tissue were detected by immunohistochemistry method.Results Western blots showed the level of phosphorylated ERK1/2 markedly increased immediately after trauma, peaking at 5 minute after trauma, and kept a a relative high level for 72 h. Treatment with U0126 effectively prevented the activation of ERKl/2 (p<0.01). The expression of C-fosmRNA had a similar trend to phosphorylated ERK1/2 in temporal profiles, but reached top as late as 30 min after trauma. Treatment with U0126 reduced the expression of c-fos mRNA dose-dependently (P<0.05) .Conclusion ERK1/2 pathway was over- and sustained- activated in diffuse brain injury after trauma. Inhibition of this pathway by U0126 can attenuate the expression of c-fos gene, which maybe reveal new therapeutic opportunity for diffuse braininjury. Objective To investigate the effect of U0126, a selective MEK1/2 inhibitor, on expression of phosphorylated extracellular signal-regulated kinases1/2 (phospho-ERK1/2) and matrix metalloproteinase-9 (MMP-9) mRNA in traumatic diffuse brain injury (DBI) of rats. To explore the role of ERK1/2 in diffuse brain injury and the potential protective mechanism via ERK1/2 pathway inhibition. Methods 115 male Sprague-Dawley rats (320—370g weight) were randomly divided into four groups: sham operated (sham group), traumatic diffuse brain injury(DBI group), treatment with U0126 before trauma (U0126 group), vehicle treated as control (DMSO group). DBI and U0126 groups were further divided into seven subgroups at different time points after trauma (5 min, 30 min, 3 h, 12 h, 24 h, 72 h, 7 d). U0126 (including 0.05mg/kg and 0.1mg/kg dose) was administered intravenously through caudal vein 30 min before trauma. Determine and evaluate the expression of phospho-ERK1/2 and MMP-9mRNA by Western blot analyses and RT-PCR respectively. The water contained in damaged brain tissue was determined by dry-wet weight method.Results Immunoblots analyses showed the expression of phospho-ERK1/2 markedly increased after trauma, showing a peak expression at 5 minute after trauma, then following a decrease. However, high level of expression of phospho-ERK1/2 sustained to 72 h after trauma. Treatment with U0126 effectively prevented the activation of ERKl/2(p<0.01). RT-PCR showed a sustained increase in MMP-9 mRNA after trauma. U0126 reduced trauma-induced MMP-9 mRNA levels dose-dependently (p<0.05 ) . The weight of water contained in damaged brain tissue was reduced by treatment with U0126 (p<0.05). By transmission electron microscope, the protective effect of U0126 on cerebral vessels was observed. Conclusions ERK1/2 pathway was over- and sustained- activated in traumatic diffuse brain injury. Inhibition of ERK1/2 cascades by U0126 can downregulate the expression of MMP-9 and attenuate brain edema, which may reveal new therapeutic opportunities for diffuse brain injury.