Abstract
Purpose :
Bruch’s membrane is an acellular, proteinaceous membrane that regulates transport between the retinal pigmented epithelium (RPE) and vascular tissue in the retina. With age, Bruch’s membrane stiffens and thickens, which is associated with the development of age-related macular degeneration (AMD). In particular, changes in Bruch’s membrane are suggested to catalyze the development of drusen, which is an early indicator of AMD. However, progressive alterations in RPE health as Bruch’s membrane changes have not been well documented. Using a tunable model of Bruch’s membrane that is representative of various stages of aging, the relationship between Bruch’s membrane and AMD can be elucidated.
Methods :
A previously described method of using hagfish proteins to develop a tunable Bruch’s membrane model was used in this experiment. To represent Bruch’s membrane at various stages in the aging process, membrane thicknesses were increased incrementally. Primary porcine RPE cells were then grown on each membrane for 3 months with biweekly media changes. At various time points, cell health was assessed via viability staining. Additionally, Trans-epithelial Electrical Resistance and immunocytochemical staining for junctional proteins were performed as a marker of monolayer health. Cells were analyzed for signs of drusen deposits, and the composition of all deposits was analyzed using spectroscopy. Additionally, permeability across the cell-membrane complex was quantified using a FITC-Dextran permeability assay to correlate deposit formation to decreased permeability.
Results :
Cells grown in this study had decreased viability as membranes thickened. As thickness increased, permeability also increased and monolayer health decreased. Drusen formed on thickened membranes and were compared to known composition of drusen in vivo.
Conclusions :
This study further supports that age-related thickening of Bruch’s membrane influences RPE health and drusen formation. Thickened membranes led to decreased cell viability, decreased monolayer functionality, and drusen formation. These findings will allow future studies to investigate the role that each of these symptoms play in the progression of AMD.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.