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Yuji Takihara, Masaru Inatani, Hideki Hayashi, Naoki Adachi, Keiichiro Iwao, Toshihiro Inoue, Minako Iwao, Hidenobu Tanihara; Dynamic Imaging of Axonal Transport in Living Retinal Ganglion Cells In Vitro. Invest. Ophthalmol. Vis. Sci. 2011;52(6):3039-3045. doi: 10.1167/iovs.10-6435.
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© 2015 Association for Research in Vision and Ophthalmology.
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.
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