The Effect Of CRMP2 On α7nAChR And Aβ Expression After Diffuse Axonal Injury In Rats

Objective:To study the effect of CRMP2 on α7n ACh R and Aβ expression after diffuse axonal injury.Methods:1. Animal group: The rats were divided into control group, DAI group, AAV group and CRMP2 group. AAV group and CRMP2 group were injected AAV virus and CRMP2 virus,respectively,and then received diffuse axonal injury. DAI group was only injured, but not injected virus.Each injury group was put to death in the DAI 7d, 14 d and 30 d, and specimens were collected to be measured.2. Establishment of DAI rat model:A diffuse axonal injury device designed by our group was used to establish DAI rat model.3.Preparation and injection of virus: CRMP2 gene was inserted into AAV expression vector by gene recombination technology. Then together with AAV2 packaging plasmids, the recombinant plasmid AAV2CRMP2 were co-transfected into HEK393 cell to package AAV virus, and viral titer was determine. AAV group was injected 2ul empty AAV virus, and CRMP2 group was injected AAV-CRMP2 to right hippocampus of rats by a microinjector.4. Histological study: Rats in each group were put to death after deep anesthesia. Then the brain tissues were removed and fixed 24 hours after paraffin embedded. α7n ACh R and Aβin each group were detected by immunofluorescence at different time points.5. α7n ACh R and Aβexpression: Rats in each group were put to death after deep anesthesia. Then the right hippocampus was retracted. α7n ACh R and Aβm RNA were measured by Real-time PCR. Aβ protein level was detected by ELISA.6. Statistical analysis: Using SPSS 18 software for statistical analysis. P<0.05 indicated the significant difference.Results:1. Histological study:α7n ACh R and Aβwere both expressed in the hippocampus of rats. Immunofluorescence assay indicated that 7n ACh R and Aβshowed red fluorescence, and nuclei emit blue fluorescence.2.Expression of α7n ACh R in hippocampus: At the different time points after injury, compared to control,α7n ACh R protein level in DAI group and AAV group was higher, and there was a statistically significance(p<0.05). But DAI group showed no difference with AAV group. α7n ACh R in CRMP2 group was higher than AAV group, and was statistically significant.3.Expression of Aβ in hippocampus: At the different time points after injury, compared to control, Aβ in DAI group and AAV group was higher, and there was a statistically significance(p<0.05). But DAI group showed no difference with AAV group. Compared with DAI group and AAV group, Aβ in CRMP2 group was significantly reduced at 7d and 14 d after injury(p<0.05),but no difference at 30 d. The DAI group, AAV group and CRMP2 group all showed higher expression of Aβ than control group.(p<0.05)。Conclusions:1.α7n ACh R expression decreased and Aβ increased in rat hippocampus after DAI.2.Overexpression of CRMP2 increased the expression of α7n ACh R and decreased the expression of Aβ after DAI.