Comparative evaluation of D-galactose and hydrogen peroxide for neurosenescence induction: an in vitro study

The progressive accumulation of senescent neuronal cells promotes sustained inflammatory responses and has been recognised as the key contributor to age-associated neurodegenerative changes. However, the molecular mechanisms underlying neuronal senescence and its impact on neural homeostasis remain poorly defined. Therefore, robust in vitro models recapitulating neuronal senescence are required for better mechanistic insight. Chemical inducers like Hydrogen peroxide (H₂O₂) and D-galactose (D-gal) are widely used to induce senescence in cell culture systems. In the present study, we systematically compared H₂O₂ and D-galactose induced senescence in human pre-neuroblastoma SH-SY5Y cells, to establish a reliable cellular model for neuronal aging and understand its implications in brain degeneration.

Cellular senescence was assessed based on morphological alterations as well as senescence-associated markers like SA-β-gal activity, expression of senescence-associated genes and proteins such as p16, p21, p53, and γH2AX. SH-SY5Y cells exhibited altered cellular morphology and changes in senescence-associated molecular markers following treatment. H₂O₂ treatment induced senescence-associated changes within five days, whereas senescence was observed within 24 h following D-galactose exposure. Both treatments resulted in increased cellular granularity and flattened or fibroblast-like morphology, although the kinetics of senescence induction differed between the two models.

The results demonstrated that D-galactose- induced a more rapid and pronounced senescence phenotype in SH-SY5Y cells compared with hydrogen peroxide based on the molecular hallmarks of aging. Based on the study, it can be concluded that the D-galactose- induced model provides a more reliable and physiologically relevant in vitro model for investigating neuronal senescence .