Development of image analysis system for quantitative evaluation of neurite degeneration

我们构建了一个实验系统，通过在线图像分析的神经元形态定量评价在分离或器官型培养的中枢神经元，从而研究神经元变性和神经保护。(1)将DiI注射到新生大鼠的上级丘，制备视网膜组织培养物，观察视网膜神经节细胞（RGCs）的形态。RGC被鉴定为在视网膜内的一个焦平面中对齐的明亮荧光点。在培养过程中，荧光点的数量和大小逐渐减少。放线菌酮或放线菌素D显着抑制活性RGCs数量的减少。DEVD，一种选择性的半胱天冬酶-3抑制剂，也显着抑制RGC活力的下降，DNA片段被发现限制在神经节细胞层。这些结果表明，RGC活力的逐渐降低是由细胞凋亡的诱导引起的。此外，一氧化氮脱 ...更多信息 来源于神经元型一氧化氮合酶（NOS）的一氧化氮合酶（NOS）抑制剂L-NAME和相对选择性的神经元型NOS抑制剂7-硝基吲唑（7-nitroindazole）可显著抑制RGC活力的下降，从而在诱导RGC凋亡中发挥重要作用。(2)我们发现，用含有高浓度Mg^<2+>或含有NMDA受体拮抗剂的培养基培养中脑切片，可导致多巴胺能神经元数量的急剧减少。这些处理后，多巴胺能神经元的轴突也有明显的萎缩变化。同时应用腺苷酸环化酶激活剂或环腺苷酸类似物可消除慢性Mg^<2+>和NMDA拮抗剂的作用。这些结果表明，与自发神经元活动相关的NMDA受体的适度激活对中脑多巴胺能神经元的早期发育和维持做出了重大贡献，其中环AMP相关的细胞内信号传导起着关键作用。少

We constructed an experimental system for quantitative evaluation of neuronal morphology by on-line image analysis of central neurons in dissociated or organotypic cultures, thereby investigated neuronal degeneration and neuroprotection. (1) We prepared retinal tissue cultures from neonatal rats that received DiI injection into the superior colliculus, and observed morphology of retinal ganglion cells (RGCs). RGCs were identified as bright fluorescent spots aligned in one focal plane within the retina. The number and size of fluorescent spots gradually decreased during the course of cultivation. Cycloheximide or actinomycin D markedly inhibited the decrease in the number of viable RGCs. DEVD, a selective caspase-3 inhibitor, also significantly inhibited the decrease in RGC viability, and DNA fragmentation was found to be restricted in the ganglion cell layer. These results indicate that the gradual decrease in RGC viability results from induction of apoptosis. Moreover, nitric oxide de … More rived from neuronal nitric oxide synthase (NOS) was shown to play an important role in induction of apoptosis in RGCs, because an NOS inhibitor L-NAME as well as a relatively selective neuronal NOS inhibitor 7-nitroindazole markedly inhibited the decrease in RGC viability. (2) We found that cultivation of midbrain slice cultures with medium containing high concentrations of Mg^<2+> or that containing NMDA receptor antagonists resulted in a drastic decrease in the number of dopaminergic neurons. There were also prominent atrophic changes in neurites of dopaminergic neurons remaining after these treatments. Co-application of an adenylyl cyclase activator or a cyclic AMP analog abolished the effects of chronic Mg^<2+> and NMDA antagonists. These results suggest that moderate activation of NMDA receptors associated with spontaneous neuronal activity makes significant contribution to early development and maintenance of midbrain dopaminergic neurons, where cyclic AMP-related intracellular signaling plays a key role. Less