Lipid Peroxidation Increases Lens Stiffness and Is Associated With αA-Crystallin Accumulation at the Plasma Membrane: An Ex Vivo Study

Purpose: The purpose of this study was to establish a long-term ex vivo mouse lens culture system and determine whether lipid peroxidation directly drives cataract-like changes in lens transparency, light scattering, stiffness, and crystallin biochemistry. Methods: Intact lenses from 3-month-old C57BL/6J mice were isolated and cultured in M199 for up to 2 weeks. Lipid peroxidation was induced by GPX4 inhibition (RAS-selective lethal 3 [RSL3]) or glutathione depletion (buthionine sulfoximine plus dimethyl fumarate [BSO+DMF]) and, where indicated, inhibited by liproxstatin-1 (Lip-1). Lens opacity was monitored by dark-field imaging. Visible absorbance spectra and a 440 to 650 nm scattering index were quantified using a custom 3D-printed cuvette mold. Lens stiffness was measured by MicroTester LT compression. Native membrane fractions were isolated by sucrose gradients for αA-crystallin immunoblotting, and protein aggregation was assessed by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). Results: Lenses remained transparent for at least 2 weeks in culture. RSL3 or BSO/DMF induced robust lipid peroxidation (4-hydroxynonenal [4-HNE]), progressive nuclear opacity, increased 440-nm absorbance and light scattering, and significantly elevated lens stiffness. Lip-1 markedly attenuated the absorbance/scattering changes. Lipid peroxidation increased αA-crystallin membrane association and promoted high-molecular-weight protein aggregate formation. Conclusions: This ex vivo organ-culture model provides direct evidence that lipid peroxidation is sufficient to induce cataract-relevant lens stiffening and opacification, accompanied by crystallin-membrane association and protein aggregation. These findings establish lipid peroxidation as a key contributor to cataract-associated optical, mechanical, and biochemical changes.