| PartⅠ Establishment and evaluation of spinal ischemia reperfusion injurymodel in adult ratsObjective: To build an animal model of spinal ischemia reperfusion injury by inadult rats, then to observe the changes and differences in locomotor scores andhistopathological after spinal ischemia reperfusion injury.Methods: We built a spinal ischemia reperfusion injury model occlusion of thedescending thoracic aorta combination with systemic hypotension (40mmHg) in adultrats. Rats were assigned randomly to4groups(Sham Group，Ischemia8min Group，Ischemia10min Group，Ischemia12min Group,10rats ineach group). According todifferent occlusion durations, the neurologic function scores system is used to evaluatechangeof the locomotor scores24h and48h afte spinal ischemia reperfusion injury,meantime, the HE(Hematoxylin and Eosin) and the Nissl staining is used to observe thehistopathological change after spinal ischemia reperfusion injury.Results: Forty-eight hours after the infusion, the rats developed the paraplegia at60％，100％or100％in Ischemia8min Group，Ischemia10min Group，Ischemia12minGroup respectively.There was significantly haemorrhage, edema, degeneration, vacuoleand so on after spinal ischemia reperfusion injury in rats.Conclusion: Establish a spinal ischemia reperfusion injury model in adult ratssuccessfully.The best of time ischemia is10min to establish a spinal ischemiareperfusion injury model. Part Ⅱ The Expression of autophagic correlative Protein: Beclin-1, LC3and P62in lesion tissues after spinal ischemia reperfusion injury in ratsObjective: To investigate whether autophagy occurred in the spinal cord tissueafter spinal ischemia reperfusion injury. We also aimed to compare the expression valueof autophagic correlative Protein: Beclin1, LC3and P62; Beclin1, LC3positive cell indifferent time point after spinal ischemia reperfusion injury.Methods: We seperated the rats into two groups randomly:Ischemia group andSham group. Ischemia group had five time point. We built the spinal ischemiareperfusion model using the way mentioned in the Part Ⅰ, while the rats in Sham groupexperience the same operation without thoracic aorta occlusion. At every time point, weused the transmission Electron Microscope to observe the ultrastructure of spinal cordtissue in order to find the autophagosome. The Western-blot were used to measure theprotein expression of Beclin1, LC3and P62in every time point after spinal ischemiareperfusion injury. Immunohistochemical staining of LC3and beclin1, counting andcalculation of LC3and beclin1positive cells.Result: We found that the autophagosome could be seen in the neuron cell at everytime point after spinal ischemia reperfusion injury. the expression of Beclin1、LC3protein to have significantly increased on the injured lumbar from3h, peaked at24hafter spinal ischemia reperfusion injury, the expression of P62protein decreasedrespectively. The increase of LC3and Beclin1positive cells at the injured lumbarspinal cord were observed from3h, peaked at24h after spinal ischemia reperfusioninjury. The expression of LC3and Beclin1were observed in neurons, astrocytes andoligodendrocytes.Conclusion:1、 Compare to the Sham group, the autophagic correlativeprotein:Beclin1and LC3were found up-regulation from3h to48h after spinal ischemia,while peaked at24h. LC3and Beclin1expressed in neurons, astrocytes andoligodendrocytes cell. LC3and Beclin1positive cells increased at the injury spinalcord. This study suggested autophagy is associated with spinal cord ischemiareperfusion injury. PartⅢ:The influence of3-MA in autophagy after Spianl ischemia reperfusioninjury in rats.Objective：Aim to evaluate how the3-MA by Intrathecal injection, which is aaccredited autophagic inhibitor, effected on neuron cells.Methods: We separated the rats into three groups randomly, there were Shamgroup, Ischemia groups,3-MA groups. Each group had four time period, they are3H,12H,24H and24H after spinal ischemia reperfusion injury. The neurologic functionscores system is used to evaluate change of the locomotor scores after spinal ischemia24H and48H. The normal motor neurons of anterior horn were counted.TheWestern-blot were used to measure the protein expression of Beclin1, LC3and P62inevery time point after spinal ischemia reperfusion injury. Immunohistochemical stainingof LC3and beclin1, counting and calculation of LC3and beclin1positive cells.Result:3-MA groups demonstrated a significant decrease in neurologic functionaloutcome on24h and48h than ischemia groups after spinal ischemia reperfusion injury.In the four time periods we observed number of normal motor neuron was more in the3-MA groups than in the ischemia groups; expression of LC3and Beclin1protein werelower in the3-MA groups than in the ischemia groups following spinal ischemiareperfusion, of course they are also higher than in the sham group.P62was higher in3-MA groups than ischemia groups. the differences between each two groups hadsignificant difference(p<0.05). the number of LC3and Beclin1positive cells inischemia groups were significantly higher than those3-MA groups at each time point,and they had significant difference(p<0.05).Conclusion: autophagy inhibitor3-methyladenine (3-MA) attenuated the ischemicinsults, improved neurologic function and number of normal neurons; inhibited theinduction of LC3and Beclin1, reversed the reduction of p62simultaneously. inhibitionof the autophagic pathway plays a neuroprotective role against spinal ischemiareperfusion injury. |
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