Brillouin microscopic assessment of retinal stiffness during reactive Müller gliosis.

Increased retinal stiffness is associated with reactive gliosis, fibrosis, and extracellular matrix remodeling-hallmarks of early retinal damage in conditions such as age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy. In this study, we demonstrate that Brillouin microscopy can detect stiffness changes in the damaged mouse retina that coincide with reactive Müller gliosis. Using acute retinal slice cultures, we generated two-dimensional Brillouin maps of both NMDA-damaged and untreated control retinas. Raw spectral images were processed to calculate the Brillouin frequency shift (BFS), which serves as a readout of tissue stiffness. Within 24 h of NMDA treatment, damaged retinas exhibited a significant increase in BFS relative to controls, indicating a rapid rise in retinal stiffness following neuronal cell death. While this biomechanical change likely reflects multiple cellular and molecular contributions, we show that the Brillouin signal localizes within the radial domain of Müller glia. These findings, along with single-cell mRNA sequencing analysis of damaged retinas, suggest that reactive Müller glia are key contributors to the observed increase in retinal stiffness. Collectively, our results establish Brillouin microscopy as a powerful tool to spatially resolve stiffness changes in the retina during neurodegeneration, provide a rationale for probing the specific mechanisms underlying these biomechanical alterations, and support the further development of Brillouin imaging as an in vivo platform for detecting early retinal pathology.