Abstract
Purpose :
To investigate autophagy activity in the outflow pathway and retina tissues in the aging and experimental ocular hypertensive mouse model.
Methods :
Young (4 m.o.) and old (18 m.o.) C57BL/6J and GFP-LC3 mice, which express the autophagosome marker LC3 fused to GFP, were subjected to unilateral injection of hypertonic saline into a limbal vein. Up to a total of three injections, separated two weeks apart, were performed as necessary. Non-injected eyes served as control and for aging studies. IOP was measured on a weekly basis using a rebound tonometer. Tissues used for WB and qPCR analysis were immediately processed for protein and mRNA isolation, respectively. Protein expression levels of LC3B, Lamp1, and p62 were evaluated by WB and/or immunofluorescence. mRNA expression levels of Atg genes were quantified by qPCR. RGC count was performed in whole-flat mounts of neuroretinas using an anti-Brn3a antibody. Optic nerves were fixed with 4% PFA and resin-embedded for axon counts and electron microscopy.
Results :
In contrast to old mice, which developed sustained elevated IOP with a single injection (C57BL/6J: 89%, ΔIOP: 292.6 ± 311.3, n=18; GFP-LC3: 94%, ΔIOP: 309.1± 279.6, n=17), just a small fraction of the mice in the young group developed modest elevation of IOP (C57BL/6J: 27%, ΔIOP: -15.92 ± 135.4 mmHg, n=26; GFP-LC3: 40%, ΔIOP: 73.12 ± 253, n=17), even after a total of three injections. Interestingly, both the percentage of animals that developed elevated IOP and the mean ΔIOP were significantly higher in the transgenic compared to C57BL/6J mice (p=0.005, t-test). IF and WB analysis showed higher LC3-II and lower p62 levels in the iridocorneal (p<0.0001) and retina (p=0.02) tissues from aged mice compared to young mice. Moreover, LC3-II levels correlated with IOP (r=82.79%, p=0.06). qPCR analysis showed downregulated expression of mTOR in the aging mice. As expected, injected OHT eyes displayed axonal degeneration and RGC death. RGC and axon loss was significantly exacerbated in aging, especially when in combination with GFP-LC3 transgene expression (RGC count: p=0.02; axon count: p=0.003, n=6). Autophagic figures were observed by EM in the degenerating axons.
Conclusions :
All together, our results indicate that autophagy is activated in the TM and retina tissues with aging, and that this activation of autophagy predisposes IOP elevation in response to insult and subsequent neurodegeneration in experimental glaucoma.
This is a 2020 ARVO Annual Meeting abstract.