利用双光子显微镜在体观察脊髓半切后小鼠感觉皮层后肢代表区树突棘的变化

发布时间：2018-04-20 04:32

本文选题：双光子成像 + 树突棘　；参考：《中国人民解放军医学院》2015年硕士论文

【摘要】：目的脊髓损伤可以导致多层次的中枢神经系统的重塑,其中就包括大脑皮层。本实验使用双光子显微镜活体观察的方法探究脊髓半切后小鼠大脑双侧后肢感觉和运动皮层树突棘和轴突膨体的变化以探讨脊髓损伤后皮层的重塑问题。方法将40只YFP小鼠随机分为两大组：感觉皮层组和运动皮层组,每组20只,每大组再随机分为两小组：对照组和脊髓损伤组(SCI组),每组10只,对照组切除椎板不损伤脊髓,脊髓损伤组切除椎板同时半切左侧胸段脊髓(T12)。分别在脊髓损伤前1天、损伤后3天(3d)、2周(2w)和1月(1m)时对两侧后肢感觉皮层(感觉皮层组)和运动皮层(运动皮层组)区域各成像四次。通过对同一位置的同一树突棘或轴突膨体(运动皮层)在不同时间点图像的对比,统计出新形成、消失和稳定存在的树突棘或轴突膨体(运动皮层)的数量,计算变化的概率以及和时间的关系,同时计算出稳定、消失、新形成的树突棘在以后较长时间段的存活率。结果感觉皮层重塑方面,与对照组相比,SCI组半切后3d和2w时对侧感觉皮层新形成的树突棘明显增加,双侧感觉皮层在脊髓半切后2w和1m时消失的树突棘明显增多,稳定的树突棘数量明显减少。在损伤后3d到2w期间,SCI组双侧皮层的原本稳定的树突棘的存活率明显下降。与SCI组对侧皮层和对照组相比,SCI组同侧皮层消失的树突棘重新出现的概率下降。在损伤后2w到1m期间,SCI组双侧皮层新形成的树突棘的存活率下降。运动皮层重塑方面,与对照组相比,SCI组半切后在1个月内两侧运动皮层轴突膨体并无明显变化;而在树突棘方面,与对照组相比,SCI组双侧运动皮层在脊髓半切后2w时消失的树突棘明显增多,稳定的树突棘数量明显减少,半切后1m时对侧运动皮层新形成的树突棘明显增加。在损伤后3d到2w期间,SCI组双侧运动皮层的原本稳定的树突棘的存活率明显下降,消失的树突棘重新出现的概率下降。在损伤后2w到lm期间,SCI组对侧运动皮层新形成的树突棘的存活率下降。结论脊髓半切后小鼠后肢感觉和运动皮层均发生重塑,重塑发生在两侧皮层。重塑不仅涉及树突棘的减少,也存在大量新形成的树突棘。对于运动皮层来说,皮层中突触的重塑主要发生在突触后组成部分树突棘而非突触前组成部分轴突膨体。在脊髓损伤后前期,感觉皮层重塑主要发生在半切对侧皮层,以树突棘新形成为主;其后重塑便涉及双侧皮层,以树突棘消失为主。脊髓损伤后,感觉和运动皮层以往稳定的树突棘不再稳定,消失后的树突棘的重新出现的概率下降,新形成的树突棘的存活率同样下降。
[Abstract]:Objective Spinal cord injury can lead to multiple levels of central nervous system remodeling, including the cerebral cortex. In order to explore the remodeling of cortex after spinal cord injury (sci), the changes of dendritic spine and axonal bulge in bilateral posterior limb and motor cortex of mice after spinal cord hemisectomy were investigated by using two-photon microscope in vivo. Methods 40 YFP mice were randomly divided into two groups: sensory cortex group (n = 20) and motor cortex group (n = 20). Each group was randomly divided into two groups: control group (n = 10) and spinal cord injury group (n = 10). In the spinal cord injury group, the vertebral lamina was excised and the left thoracic spinal cord was cut half. The sensory cortex (sensory cortex group) and motor cortex (motor cortex group) of bilateral hind limbs were respectively imaged four times before 1 day, 3 days after injury for 2 weeks and 1 month for 1 m. By comparing the images of the same dendritic spine or axonal bulge (motor cortex) in the same position at different time points, the number of newly formed, disappeared and stable dendritic spine or axocele (motor cortex) was calculated. The probability of change and the relationship between time and change were calculated, and the survival rate of the newly formed dendritic spine in a longer period of time was calculated. Results in the sensory cortex remodeling, compared with the control group, the newly formed dendritic spine in contralateral sensory cortex increased significantly 3 days and 2 weeks after hemisection in sci group, and the dendritic spine disappeared in bilateral sensory cortex at 2 weeks and 1 m after spinal cord hemisection. The number of stable dendritic spine decreased significantly. The survival rate of the previously stable dendritic spine in bilateral cortex of sci group decreased significantly between 3 days and 2 weeks after injury. Compared with the contralateral cortex of SCI group and control group, the probability of reappearance of dendritic spine disappeared in ipsilateral cortex of sci group was decreased. The survival rate of the newly formed dendritic spine in sci group decreased from 2 weeks to 1 m after injury. In motor cortex remodeling, there was no significant change in axonal bulge of bilateral motor cortex in sci group within 1 month after hemisection compared with control group, but in dendritic spine, there was no significant change in axonal bulge in bilateral motor cortex in sci group. Compared with the control group, the number of dendritic spine disappeared and the number of stable dendritic spine decreased significantly at 2 weeks after spinal cord hemisection in sci group, and the newly formed dendritic spine in contralateral motor cortex increased significantly at 1 m after hemisection. The survival rate of the previously stable dendritic spine in the bilateral motor cortex of the sci group decreased significantly from 3 days to 2 weeks after injury, and the probability of reappearance of the disappeared dendritic spine decreased. The survival rate of the newly formed dendritic spine in the contralateral motor cortex decreased between 2 weeks and 1 m after injury in the sci group. Conclusion the sensory and motor cortex of the hind limb were remodeled after hemisection of spinal cord in mice, and the remodeling occurred in the bilateral cortex. Remodeling involves not only the reduction of dendritic spine, but also a large number of newly formed dendritic spine. For motor cortex, synaptic remodeling occurs mainly in postsynaptic dendritic spine rather than presynaptic axonocele. In the early stage of spinal cord injury, sensory cortex remodeling mainly occurred in the half-cut contralateral cortex, mainly as dendritic spine, and then the remodeling involved bilateral cortex, mainly the disappearance of dendritic spine. After spinal cord injury, the previously stable dendritic spine of sensory and motor cortex was no longer stable, and the probability of reappearance of the disappeared dendritic spine was decreased, and the survival rate of newly formed dendritic spine was also decreased.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R651.2

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