Purchase this article with an account.
Nishant Rajiv Sinha, Ratnakar Tripathi, Praveen Kumar Balne, Sydney L. Green, Riley M. Laub, Rajiv R Mohan; mTORC regulation of autophagy in the corneal stroma following chemical injury. Invest. Ophthalmol. Vis. Sci. 2022;63(7):113 – A0211.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Cornea is susceptible to accidental/intentional chemical injury. Chlorine (Cl2) exposure to the eye causes corneal damage, redness, tearing, itching, and blurred vision. Autophagy and lysosomal biogenesis play a vital role in corneal homeostasis, repair, and transparency maintenance. This study tested if mTORC1-mediated dysfunctional autophagy and lysosomal biogenesis is associated with Cl2 induced corneal damage.
Primary human stromal fibroblast cultures (hCSF) and ex vivo organ culture model using donor human corneas were used. hCSF cultures were exposed to +/- Cl2 (0.5ppm liberated from 0.001% NaOCl) for 72h (n=6). Thereafter, cultures were fixed in 4% paraformaldehyde and generating protein lysates and mRNA and cDNA. The changes in autophagosomal and lysosomal signature genes (ATGs, LC3, SQSTM1/p62, LAMP1, mTORC1, and TFEB) were measured with qRT-PCR and proteins levels and distribution with western blotting and/or immunofluorescence. Ex vivo human corneas were exposed to 0.5ppm Cl2 for 30 minutes and incubated for 2 months (n=3). mRNA and protein levels of autophagy markers (LC3, Beclin1, and p62) were analyzed with qRT-PCR, western blotting, and immunofluorescence.
hCSF exposed to Cl2 had decreased phosphorylated mTORC compared to vehicle. Cl2 exposure to hCSF significantly increased mRNA and protein levels of LC3 (p=<0.01, p=<0.01), Beclin1 (p=<0.01, p=<0.01), and p62 (p=<0.01, p=<0.01) compared to vehicle. ICC of hCSF exposed to Cl2 had higher levels of LC3 and Beclin1 with increased levels and accumulation of p62. Further, ICC showed TFEB distribution is greater in cytosol when exposed to Cl2 compared to vehicle. LAMP1 is decreased in Cl2 exposed hCSFs. IHC of ex vivo cultures showed increased protein levels of LC3, Beclin1, and p62 after Cl2 exposure compared to vehicle.
Autophagy mechanism is involved in Cl2 mediated toxicity to the cornea. Further ongoing studies are expected to offer additional mechanistic information regarding the role of autophagy in corneal injury, repair and homeostasis.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
This PDF is available to Subscribers Only