Large-volume ultrastructural analysis of photoreceptor neurons in retinitis pigmentosa mouse models by improved expansion microscopy

Abstract

Purpose : Ultrastructural analysis is crucial for comprehending photoreceptor cell biology. Electron microscopy has traditionally been employed for this purpose, but its utility is constrained by small sample sizes and limited scalability. To address this limitation, expansion microscopy has been improved to accommodate optical super-resolution imaging of larger volumes. Our goal was to enhance the utilization of expansion microscopy for investigating the mouse retina, particularly concentrating on photoreceptors. Additionally, we aimed to acquire a deeper understanding of the ultrastructural alterations linked to inherited retinal degeneration.

Methods : We used wild-type (WT) mouse cryo retinal sections to optimize the expansion microscopy methods. Then, optimized conditions were utilized to analyze ultrastructure of photoreceptor cells in the mouse models of retinitis pigmentosa (Rho^P23H/P23H and Rho^Q344X/Q344X). As controls, retinas from age-matched WT mice were studied.

Results : We found that conventional expansion microscopy is not suitable for retina research due to its tendency to fragment the retina. However, we have developed an advanced expansion microscopy technique that successfully expands the retina without fragmentation and is compatible with post-expansion antibody labeling. We were able to statistically compare ciliary structures in the retina among different genetic backgrounds. At postnatal day 11 (PND11), a stage characterized by minimal rod cell death in both Rho^P23H/P23H and Rho^Q344X/Q344X mice, we observed that the density of photoreceptor cilia in Rho^P23H/P23H mice had decreased to 50-60% when compared to the WT. Conversely, the density in Rho^Q344X/Q344X mice remained similar to that of the WT group. The subcellular localization of mutant rhodopsin and the structure of rod outer segments also showed distinct differences between Rho^P23H/P23H and Rho^Q344X/Q344X mice.

Conclusions : The novel technique enabled us to conduct statistical analyses on ultrastructures within mouse retinal sections. Through the examination of photoreceptor cilia and their associated structures, we pinpointed statistically significant alterations in photoreceptor cell ultrastructures. Importantly, this technique holds the potential for application in other neuronal tissues characterized by diverse cell layers.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.