Abstract
Purpose :
The etiology of age-related macular degeneration (AMD) is multifactorial and the exact mechanisms of outer retinal changes in AMD remains to be elucidated. Previous studies have shown that abnormal activation of mammalian target of rapamycin complex 1 (mTORC1) caused by the deletion of the mTORC1 upstream inhibitor tuberous sclerosis complex I (TSC1) leads to the development of AMD-like retinal changes in mice. This study aims to determine the changes in RPE monolayer morphology in the TSC1 AMD model using the REShAPE software tool.
Methods :
Mice with conditional deletion of TSC1 (cKO) from both rod and cone photoreceptors were aged to 12 months before enucleation and flat-mounting of eye cups. Eleven (5 wild type, 6 cKO) mouse eyes were analyzed in total. Freshly enucleated mouse eyes were fixed in 4% paraformaldehyde solution in TRIS-buffered saline and dissected. RPE cells were stained with Phalloidin or ZO-1 to outline cell junctions, flat-mounted, and imaged using a Leica DM6000 microscope under 20x magnification. Images were processed by REShAPE, a software-based morphometric analysis program, to assess RPE cell shape, size, and number of neighboring cells. Optimization was achieved by averaging, height color coding, maximum projection, and transparency settings to find the best method for producing high-quality images for analysis with REShAPE.
Results :
High quality images for morphometric analysis by ReShAPE were generated by thorough removal of extraneous connective tissue during dissection, use of Phalloidin to stain cell junctions, and use of post-processing of Z-stack images. Phalloidin (1:80 dilution) was found to stain the perimeter of RPE cells optimally and performed better than ZO-1, resulting in higher quality images with data brightness and resolution of cell junctions. REShAPE processing will generate data to determine RPE cell shape (hexagonality, perimeter, etc.), area, and number of neighboring cells between wildtype and TSC1 cKO mice.
Conclusions :
We developed methods to optimize tissue processing and staining for high quality images that are critical for determining morphological characteristics of normal and diseased RPE cells in a mouse AMD model. This method is expected to be important for assessing the efficacy of novel therapeutics that combat AMD.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.