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Rong-Kung Tsai, Yao-Tseng Wen; The roles of autophagy and mTOR in the neuro-protection of optic nerve crush models in rats. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3871.
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© ARVO (1962-2015); The Authors (2016-present)
To illustrate the role of autophagy machinery in optic nerve (ON) injury, two autophagy activators, rapamycin and p62 siRNA, were used to evaluate their neuroprotective effects in a rat model of ON crush .
Adult male Wistar rats immediately received an intravitreal injection of p62 siRNA or intravitreal injections of rapamycin after ON crush experiments. Two weeks after ON crush, the neuroprotective effects were evaluated by measuring flash visual-evoked potentials (fVEP) and RGC survival rate. The anti-apoptotic effect was measured by TUNEL assay in the retinal sections. The inflammatory condition in the ON was determined by counting the number of ED1-positive cells. The level of autophagy-related markers was examined by immunoblotting and immunostaining analysis.
The P1-N2 amplitudes of fVEP were found significantly higher in the p62 siRNA-treated group compared with that of the rapamycin-treated group and the ON crush group (41.5±10.5 vs. 25.2±9.8 and 14.6±11.6, p<0.05). The RGC densities of central retina and mid-peripheral retina were statistically higher in the p62 siRNA-treated group than that of the rapamycin-treated groupand the crush group (center: 1282±148; 573±144 and 636±160 respectively, mid-periphery: 882±148, 460±96 and 462±230 respctively; p<0.05). The number of apoptotic RGC was compatible with RGC densities by reducing 2.26- and 2.34-fold of p62 siRNA treatment as comparing with the rapamycin and the scramble siRNA in ON-crush rats. Macrophage infiltration at the ONs were statistically lower in the p62 siRNA-treated group compared with the rapamycin-treated group and the ON crush group (86±40 vs. 277.0±154.6 and 301.3±115.4, p<0.05). Both immunoblotting and immunostaining indicated that intravitreal injections of p62 siRNA and rapamycin significantly elevated the expression of LC3 II and LAMP1 and reduced P62 in contrast to scramble siRNA. However, rapamycin significantly reduced 4.2-fold of retinal mTOR expression compared with treatment with p62-siRNA in the ON crush model (p<0.05).
Conclusions:Intravitreal injection of p62 siRNA and rapamycin can activate autophagy after ON crush. However, intravitreal injections of rapamycin also down-regulate the m-TOR expression. This crucial mechanism explains the difference of results between p62 siRNA and rapamycin in the rat ON crush model.
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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