The Expression And Effect Of Autophagy After Spinal Cord Injury In Rats: An Experimental Study

PartⅠEstablishment And Evaluation of Acute Spinal Cord Injury Model in Adult RatsObjective: To build an animal model of spinal cord injury by clamp force in adult rats, then to observe the changes and differences in locomotor scores and histopathological after spinal cord injury(SCI) in the three groups: Group Sham.Group SCI and Group 3-MA.Methods: Build a spinal cord injury model in dorsal transsection model (T8) by clamp force,The Basso-Beattie-Bresnahan (BBB) scores system is used to evaluate change of the locomotor scores after SCI, meantime, the HE(Hematoxylin and Eosin) staining and the Nissl staining are used to observer the histopathological change after the SCI.Results: There was significantly haemorrhage, edema, degeneration, vacuole and so on after spinal cord injury in rats with T8 dorsal transaction .The BBB scores of rats have no significant difference between Group SCI and Group 3-MA after SCI(p>0.05).The histopathological scores has significant difference between Group SCI and Group 3-MA after SCI (p<0.05)Conclusion: 1,Establish a spinal cord injury model in adult rats successfully .2.The histopathological changes of Group 3-MA is more than the Group SCI at the time point of 3 days after the operation。 PartⅡThe Expression of autophagic genes: Beclin-1 and MAP1-LC3B in lesion tissues after spinal cord injury in ratsObjective: TO investigate whether autophagy occurred in the spinal cord tissue after spinal cord injury(SCI).We also aimed to compare the expression value of autophagic correlative gene and Protein: Beclin1 & LC3B in different time point after SCI.Methods: Seperated the rats into two groups randomly :SCI group and control group. Each group had five time point. We built the SCI model using the way mentioned in the PartⅠ,while the rats in control group experience the same operation without injurying the spinal cord. At every time point, we used the transmission Electron Microscope to observe the ultrastructure of spinal cord tissue in order to find the autophagosome. The mRNA expression of Beclin1 and LC3B were also detected by the RT-PCR technique. The Immune-Histochemical(IHC) and Western-blot were used to measure the protein expression of Beclin1 and LC3B in every time point after SCI.Result: We found that the autophagosome could be seen in both the Neuron cell and the Glial cell at every time point after SCI. The mRNA expression value of Beclin and LC3B were raised since 8H Post-SCI and peaked at 3D post-SCI, and they could be detected until 21D after SCI. The protein's expression curve of Beclin1 and LC3B were the same as the gene's .Conclusion: The autophagic cell death could be detected in spinal cord injury region, both the neuron cell and glial cell experienced autophagic cell death. Compare to the contro group, the autophagic correlative gene and protein:Beclin1 and LC3B were found up-regulation from 8H to 21D after SCI, while peaked at 3D post-SCI. PartⅢ:The influence of 3-MA in autophagy and apoptosis after Spianl cord injury in rats.Objective:Aim to evaluate how the 3-MA ,which is a accredited autophagic inhibitor, effected the genic and proteinic expression of Beclin1 and LC3B. Meanwhile we wished to investigate the effect of cell apoptosis after spinal cord injury by the 3-MA.Methods: We separated the rats into three groups randomly, there were Group control, Group SCI, Group 3-MA. Each group had five time period, they are 8H.1D.3D.7D.21D after SCI. In this time point, We used the SYBR GREEN RT-PCR technique to measure the mRNA's differences of Beclin1 and LC3B within the three groups. The proteinic differences of Beclin1 and LC3B within this three groups were evaluated by the Fluorescence immunohistochemistry. The level of apoptosis after SCI in the three groups were measured by the TUNEL. DNA ladder. The expression of Bcl-2 and Bax, which are closely interrelated with apoptosis, were tested by the Western-blot.Result: In the five time periods we observed, the genic expression of Beclin1 and LC3B were lower in the group 3-MA than in the group SCI, of course they are also higher than in the group Control. Also the differences between each two groups had significant difference(p<0.05). The proteinic expression of Beclin1 and LC3B in Group 3-MA were also lower than it in Group SCI, and they had significant difference(p<0.05). We found the apoptosis after SCI, and the TUNEL positive cell were much more in group 3-MA than it in Group SCI, the differences had statistical significance(p<0.05).The degree of DNA-Ladder were higher in Group 3-MA than in Group SCI. The proteinic expression of Bcl-2 is highest in group control, higher in group SCI and lowest in the group 3-MA. The difference in groups had significant difference(p<0.05). The proteinic expression of Bax is highest in group 3-MA, higher in SCI and lowest in group control. The difference in groups had significant difference(p<0.05).Conclusion: 3-MA as a accredited autophagic inhibitor, could both inhibited the ?genic and proteinic expression of Beclin1 and LC3B. Meanwhile ,the 3-MA could aggravate the apoptosis after SCI in rats by the way of inhibiting autophagy. So the autophagy could recognized as a mechanism of cellular self-protect, and autophagy played a antagonistic effect against apoptosis.