大鼠脊髓缺血再灌注损伤后自噬表达及作用的实验研究
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摘要
第一部分大鼠脊髓缺血再灌注损伤动物模型的建立与评价
目的：建立大鼠脊髓缺血再灌注损伤模型，观察不同缺血时间对脊髓缺血再灌注损伤后病理组织学和神经功能评分的影响。
方法：构建大鼠胸主动脉球囊阻断复合体循环低血压（MAP=40mmHg)脊髓缺血再灌注模型，按不同的阻断时间随机分为假手术组、缺血8min组、10min组和12min组，评定再灌注24h和48h后神经功能；观察再灌注48h后脊髓形态学变化。
结果：再灌注48h后，缺血8min、10min和12min组出现截瘫率分别为60％、100％和100％，各组比较差异有统计学意义(P<0.05)。再灌注48h后，脊髓组织出现神经细胞肿胀、坏死等改变，且随阻断时间的延长，损伤程度逐渐加重。
结论成功建立大鼠胸主动脉球囊阻断复合体循环低血压脊髓缺血再灌注模型。脊髓缺血10min是模型建立的最佳时间。
第二部分细胞自噬及其相关蛋白Beclin-1，LC3和P62在大鼠脊髓缺血再灌注损伤组织中的表达
目的：观察大鼠脊髓缺血再灌注损伤后损伤区细胞自噬是否存在，比较不同时间点细胞自噬的表达情况。
方法：将大鼠随机分为两组，假手术组和缺血组，缺血组再分五个时间点：3H，6H，12H，24H，48H。损伤组按照第一部分的方法制作脊髓缺血再灌注损伤模型，假手术组只置入导管而不打开球囊损伤脊髓，按照上述时间点处死动物后，用透射电镜观察细胞自噬，用荧光双染法检测LC3及Beclin1在各时间点的表达和在各类脊髓组织细胞中的分布，Western blot法检测自噬相关蛋白Beclin1，LC3和P62的表达情况。
结果：透射电镜发现脊髓损伤后的各个时间点，在损伤的神经元细胞中可以发现自噬囊体和自噬溶酶体明显增加，脊髓损伤后3h开始，细胞自噬相关蛋白Beclin1和LC3表达即较对照组明显上调，并且随着时间持续升高，在伤后24h时达到高峰，伤后48h有回落，但仍高于基础值，而自噬相关蛋白P62表达下调表达，和Beclin1，LC3反相同步。缺血再灌注组Beclin1和LC3阳性细胞数在各时间点明显高于假手术组（p﹤0.05），趋势与蛋白变化相符。脊髓神经元细胞，胶质细胞，小胶质细胞中均有Beclin1和LC3表达。
结论：1、脊髓缺血再灌注损伤中3H自噬表达明显上调，24H到达高峰，48H下降。神经元细胞、胶质细胞和小胶质细胞均可见自噬表达。自噬参与脊髓缺血再灌注损伤过程。
第三部分自噬抑制剂3-MA对大鼠脊髓损伤后神经细胞自噬的影响
目的：通过鞘内注射自噬抑制剂3-MA，观察自噬被抑制后对大鼠脊髓缺血再灌注损伤后神经细胞自噬的影响。
方法：将大鼠随机分成三组：假手术组，单纯缺血损伤组，3-MA组，每组分为伤后3H，12H，24H，48H各4个时间点，神经功能评分评价24h和48h小时后双下肢神经功能，脊髓前角神经元计数，Western blot法检测自噬相关蛋白Beclin1，LC3和P62检测各组各时间点蛋白表达的变化，组织荧光免疫法检测各组Beclin1和LC3阳性细胞变化。
结果：24H和48H后3-MA干预组较单纯缺血组神经功能评分显著改善（P＜0.05），在观测的的4个时间点上，3-MA治疗组脊髓前角神经元数明显多于单纯缺血组， Beclin1和LC3的蛋白上调表达和P62表达下调较单纯缺血组明显降低，差异有统计学意义（P＜0.05）。3-MA组Beclin1和LC3的阳性细胞数明显少于缺血损伤组，组间差异有统计学意义（P＜0.05）。
结论：细胞自噬抑制剂3-MA可以明显改善大鼠脊髓缺血再灌注损伤后神经功能，增加神经元存活数，抑制自噬相关蛋白Beclin1、LC3和P62的表达。减少Beclin1和LC3阳性细胞数，抑制自噬具有神经保护作用。
PartⅠ Establishment and evaluation of spinal ischemia reperfusion injurymodel in adult rats
Objective: 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 rats
Objective: 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.
目的：建立大鼠脊髓缺血再灌注损伤模型，观察不同缺血时间对脊髓缺血再灌注损伤后病理组织学和神经功能评分的影响。
方法：构建大鼠胸主动脉球囊阻断复合体循环低血压（MAP=40mmHg)脊髓缺血再灌注模型，按不同的阻断时间随机分为假手术组、缺血8min组、10min组和12min组，评定再灌注24h和48h后神经功能；观察再灌注48h后脊髓形态学变化。
结果：再灌注48h后，缺血8min、10min和12min组出现截瘫率分别为60％、100％和100％，各组比较差异有统计学意义(P<0.05)。再灌注48h后，脊髓组织出现神经细胞肿胀、坏死等改变，且随阻断时间的延长，损伤程度逐渐加重。
结论成功建立大鼠胸主动脉球囊阻断复合体循环低血压脊髓缺血再灌注模型。脊髓缺血10min是模型建立的最佳时间。
第二部分细胞自噬及其相关蛋白Beclin-1，LC3和P62在大鼠脊髓缺血再灌注损伤组织中的表达
目的：观察大鼠脊髓缺血再灌注损伤后损伤区细胞自噬是否存在，比较不同时间点细胞自噬的表达情况。
方法：将大鼠随机分为两组，假手术组和缺血组，缺血组再分五个时间点：3H，6H，12H，24H，48H。损伤组按照第一部分的方法制作脊髓缺血再灌注损伤模型，假手术组只置入导管而不打开球囊损伤脊髓，按照上述时间点处死动物后，用透射电镜观察细胞自噬，用荧光双染法检测LC3及Beclin1在各时间点的表达和在各类脊髓组织细胞中的分布，Western blot法检测自噬相关蛋白Beclin1，LC3和P62的表达情况。
结果：透射电镜发现脊髓损伤后的各个时间点，在损伤的神经元细胞中可以发现自噬囊体和自噬溶酶体明显增加，脊髓损伤后3h开始，细胞自噬相关蛋白Beclin1和LC3表达即较对照组明显上调，并且随着时间持续升高，在伤后24h时达到高峰，伤后48h有回落，但仍高于基础值，而自噬相关蛋白P62表达下调表达，和Beclin1，LC3反相同步。缺血再灌注组Beclin1和LC3阳性细胞数在各时间点明显高于假手术组（p﹤0.05），趋势与蛋白变化相符。脊髓神经元细胞，胶质细胞，小胶质细胞中均有Beclin1和LC3表达。
结论：1、脊髓缺血再灌注损伤中3H自噬表达明显上调，24H到达高峰，48H下降。神经元细胞、胶质细胞和小胶质细胞均可见自噬表达。自噬参与脊髓缺血再灌注损伤过程。
第三部分自噬抑制剂3-MA对大鼠脊髓损伤后神经细胞自噬的影响
目的：通过鞘内注射自噬抑制剂3-MA，观察自噬被抑制后对大鼠脊髓缺血再灌注损伤后神经细胞自噬的影响。
方法：将大鼠随机分成三组：假手术组，单纯缺血损伤组，3-MA组，每组分为伤后3H，12H，24H，48H各4个时间点，神经功能评分评价24h和48h小时后双下肢神经功能，脊髓前角神经元计数，Western blot法检测自噬相关蛋白Beclin1，LC3和P62检测各组各时间点蛋白表达的变化，组织荧光免疫法检测各组Beclin1和LC3阳性细胞变化。
结果：24H和48H后3-MA干预组较单纯缺血组神经功能评分显著改善（P＜0.05），在观测的的4个时间点上，3-MA治疗组脊髓前角神经元数明显多于单纯缺血组， Beclin1和LC3的蛋白上调表达和P62表达下调较单纯缺血组明显降低，差异有统计学意义（P＜0.05）。3-MA组Beclin1和LC3的阳性细胞数明显少于缺血损伤组，组间差异有统计学意义（P＜0.05）。
结论：细胞自噬抑制剂3-MA可以明显改善大鼠脊髓缺血再灌注损伤后神经功能，增加神经元存活数，抑制自噬相关蛋白Beclin1、LC3和P62的表达。减少Beclin1和LC3阳性细胞数，抑制自噬具有神经保护作用。
PartⅠ Establishment and evaluation of spinal ischemia reperfusion injurymodel in adult rats
Objective: 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 rats
Objective: 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.
引文
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