AFM-derived cytoskeletal mechanics reveal ethanol-induced injury and vitamin C-mediated repair in SH-SY5Y cells.

Oxidative stress and cytoskeleton-associated remodeling have been widely implicated, yet most current approaches to assess the cytoskeleton-associated organization and mechanics rely on fluorescent labeling or electron microscopy, which are invasive and difficult to quantify dynamically. Herein, we combine ethanol-induced injury and VC post-treatment in SH-SY5Y cells with atomic force microscopy (AFM) and image-based analysis to establish a label-free, mechanics-based cytoskeleton-associated mechanical phenotype. SH-SY5Y cells were exposed to 6% ethanol for 4 h to induce a sub-lethal injury phenotype, followed by 24 h treatment with VC (40-160 µM). Cell viability (MTT), scanning electron microscopy (SEM), and AFM quantitative imaging were used to evaluate injury and repair. Ethanol reduced the cell viability to approximately 50% and induced marked shrinkage and loss of pseudopodia, whereas 160 µM VC restored the viability to near control levels and largely recovered normal morphology. AFM maps of height, adhesion and Young's modulus revealed ethanol-induced alterations in whole-cell mechanics that were shifted toward control with increasing VC concentration. We further developed an AFM-based pipeline to derive cytoskeleton-associated maps and segmented regions from multiparametric AFM data. Quantitative analysis showed that cytoskeletal mechanics were more sensitive than whole-cell averages to both ethanol injury and VC rescue. These findings suggest that the AFM-derived cytoskeleton-associated mechanical results provide a label-free, segmentation-ready metric for assessing injury-recovery phenotypes in neuronal-lineage cells at the single-cell level, with potential utility for screening candidate protective interventions.