Dynamic imaging of axonal transport in living retinal ganglion cells in vitro.
Dynamic imaging of axonal transport in living retinal ganglion cells in vitro.
复制标题
体外活体视网膜神经节细胞轴突运输的动态成像。
DOI：
10.1167/iovs.10-6435
复制
发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Tanihara H.
中科院分区：
文献类型：
--
作者：
Takihara Y;Inatani M;Hayashi H;Adachi N;Iwao K;Inoue T;Iwao M;Tanihara H.
目的：神经营养因子轴突转运的破坏在青光眼视神经病变视网膜神经节细胞（RGCs）凋亡中起着关键作用。本研究采用活细胞体外成像技术，观察和评价脑源性神经营养因子（BDNF）在活的和轴突损伤的RGC中的轴突转运。通过两步免疫淘洗法纯化大鼠RGCs。用编码带有绿色荧光蛋白（GFP）标记的BDNF的质粒转染培养的RGCs，并通过延时成像分析BDNF-GFP在轴突和树突中的动态。在用1 mM秋水仙碱处理后检查BDNF-GFP轴突运输的变化，并且用乙锭同二聚体-1评估处理后的RGC死亡。BDNF-GFP在培养的RGCs的轴突和树突中呈囊泡状表达。延时成像显示BDNF-GFP在轴突中的平均速度（0.86±0.37 μm/s，最大值为2.03 μm/s）显著大于树突中的平均速度（0.49±0.19 μm/s，P< 0.0001）。秋水仙碱在处理后2和3小时显著抑制BDNF-GFP的轴突运输（分别为P= 0.003和0.0002），而不影响细胞活力;然而，在处理后24小时检测到RGC死亡。活细胞成像揭示了BDNF在活RGCs中轴突运输的动力学，这与树突中的运动明显不同。此外，这是可能的，以确认在细胞死亡之前，在秋水仙素处理的RGCs的轴突运输的中断。
Purpose.: Disruption of the axonal transport of neurotrophic factors plays a critical role in the apoptosis of retinal ganglion cells (RGCs) in glaucomatous optic neuropathy. Live-cell imaging in vitro was used in this study, to visualize and evaluate the axonal transport of brain-derived neurotrophic factor (BDNF) in both living and axon-damaged RGCs.Methods.: Rat RGCs were purified by a two-step immunopanning method. Cultivated RGCs were transfected with a plasmid encoding BDNF tagged with green fluorescent protein (GFP), and the dynamics of BDNF-GFP in the axons and dendrites were analyzed by time-lapse imaging. Changes in the axonal transport of BDNF-GFP were examined after treatment with 1 mM colchicine, and RGC death after treatment was evaluated with ethidium homodimer-1.Results.: The expression of BDNF-GFP showed a vesicular pattern in the axons and dendrites of cultivated RGCs. Time-lapse imaging revealed that the mean velocity of BDNF-GFP in the axons (0.86±0.37 μm/s; maximum= 2.03 μm/s) was significantly greater (P< 0.0001) than that in the dendrites (mean= 0.49±0.19 μm/s). Colchicine significantly inhibited the axonal transport of BDNF-GFP at 2 and 3 hours after treatment (P= 0.003 and 0.0002, respectively) without affecting cell viability; however, RGC death was detected 24 hours after treatment.Conclusions.: Live-cell imaging revealed the dynamics of the axonal transport of BDNF in living RGCs, which clearly differed from the movements in dendrites. Furthermore, it was possible to confirm the disruption of axonal transport in colchicine-treated RGCs before cell death.