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Seiji Miyake, Yuji Takihara, Satoshi Yokota, Yoshihiro Takamura, Masaru Inatani; Effect of axonopathy on dynamics of acidic organelle in the axon of primary cultured retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2532.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma is characterized by the progressive loss of retinal ganglion cell (RGC) and its axon. However, molecular details of relationship between cell death and axonal degeneration remain poorly understood. We investigate the implication of dynamics of autophagy for RGC death under the axonal abnormality using live imaging in vitro.
RGCs were isolated from postnatal day 3 Sprague-Dawley rats by immunopanning method. After 7 days culture, acidic organelles were stained by LysoTracker. Dynamics were quantified from kymographs. To induce axonopathy, colchicine was used. Axonal transport of acidic organelles was observed at 5 time points: before, and at 6, 24, 72, and 120 hours after colchicine stimulation. Ethidium homodimer-1 (EthD-1) was used to detect cell viability.
Property of axonal transport of 27 RGCs (n = 363) was classified into 4 categories; anterograde (1.4%), retrograde (90%), stationary (8.0%), and fluttering (0.28%). At 6 hours after induction of axonopathy for 14 (n = 236) of 27 RGCs, movement of almost acidic organelles were stationary. All acidic components were completely stopped 24 hours later. At 72 hours since giving stimulation, axonal fragmentation, and shrinking and disappearance of soma were observed in 71% of RGC. Finally, remaining RGCs were positive for EthD-1. In the control (13 of 27 RGCs), transport was kept for 120 hours and EthD-1 positive RGCs were not observed.
RGC death followed by cease of axonal transport. These results suggested that functional disorder of autophagy through axonal damage is one of the causes of glaucomatous optic neuropathy.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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