Department of Ophthalmology, Duke University, Durham, North Carolina, United States.
Invest Ophthalmol Vis Sci. 2020 Aug 3;61(10):31. doi: 10.1167/iovs.61.10.31.
To investigate autophagy in the outflow pathway and ganglion cell layer in the aging and ocular hypertensive mouse.
Both 4-month-old and 18-month-old C57BL/6J and GFP-LC3 mice were subjected to unilateral injection of hypertonic saline into a limbal vein, causing sclerosis of the outflow pathway and subsequent elevation of intraocular pressure (IOP). IOP was measured on a weekly basis using a rebound tonometer. Protein expression levels of LC3B, Lamp1, and p62 were evaluated by western blot and/or immunofluorescence. Retinal ganglion cell (RGC) count was performed in whole retinal flat mounts using an anti-Brn3a antibody. Optic nerves were fixed with 4% paraformaldehyde and resin-embedded for axon counts and electron microscopy.
In contrast to 18-month-old mice, which developed sustained elevated IOP with a single injection, 4-month-old mice were refractory to high elevations of IOP. Interestingly, both the percentage of animals that developed elevated IOP and the mean ∆IOP were significantly higher in the transgenic mice compared to C57BL/6J. Immunofluorescence and western blot analysis showed dysregulated autophagy in the iridocorneal and retina tissues from 18-month-old mice compared to 4-month-old ones. Moreover, the LC3-II/LC3-I ratio correlated with IOP. As expected, injected hypertensive eyes displayed axonal degeneration and RGC death. RGC and axon loss were significantly exacerbated with aging, especially when combined with GFP-LC3 expression. Autophagic structures were observed in the degenerating axons.
Our results indicate dysregulation of autophagy in the trabecular meshwork and retinal tissues with aging and suggest that such dysregulation of autophagy contributes to neurodegeneration in glaucoma.
研究衰老和高眼压小鼠流出道和神经节细胞层中的自噬作用。
对 4 月龄和 18 月龄 C57BL/6J 和 GFP-LC3 小鼠进行单侧角膜缘静脉注射高渗盐水，导致流出道硬化和随后的眼内压（IOP）升高。每周使用回弹眼压计测量 IOP。通过 Western blot 和/或免疫荧光评估 LC3B、Lamp1 和 p62 的蛋白表达水平。使用抗 Brn3a 抗体在全视网膜平面上进行视网膜神经节细胞（RGC）计数。视神经用 4%多聚甲醛固定并用树脂包埋进行轴突计数和电子显微镜检查。
与仅注射一次即可持续升高 IOP 的 18 月龄小鼠相比，4 月龄小鼠对高眼压不敏感。有趣的是，与 C57BL/6J 相比，转基因小鼠中发展为高眼压的动物百分比和平均 ∆IOP 均显著升高。免疫荧光和 Western blot 分析显示，与 4 月龄相比，18 月龄的虹膜角膜和视网膜组织中自噬作用失调。此外，LC3-II/LC3-I 比值与 IOP 相关。正如预期的那样，注射性高血压眼显示轴突变性和 RGC 死亡。随着年龄的增长，RGC 和轴突丢失明显加重，尤其是与 GFP-LC3 表达结合时。在变性轴突中观察到自噬结构。
我们的结果表明，衰老时小梁网和视网膜组织中的自噬作用失调，并表明这种自噬作用失调有助于青光眼的神经退行性变。
