Synchronized Imaging of Hydrogen Peroxide and Hydroxyl Radical in Pyroptosis and Epilepsy

Real-time visualization of oxidative stress in living systems poses a formidable challenge in epilepsy research, primarily due to the scarcity of tools capable of selectively distinguishing structurally similar reactive oxygen species (ROS) with high specificity. Here, we present a dual-functional fluorescent probe, HH , capable of independently detecting hydrogen peroxide (H 2 O 2 ) and hydroxyl radicals (•OH) through distinct recognition mechanisms, yielding spectrally resolved signals with high sensitivity, selectivity, and biocompatibility. HH enables high-resolution, interference-free imaging of redox dynamics in both gasdermin-mediated pyroptosis and pentylenetetrazol (PTZ)-induced epileptic cell models. Furthermore, HH was successfully employed in PTZ-induced zebrafish and kainic acid (KA)-induced mouse epilepsy models, revealing elevated ROS levels under seizure-like conditions. Notably, ROS-targeted therapeutic interventions significantly modulated oxidative profiles, demonstrating HH ’s potential for therapeutic evaluation. This work not only fills a critical methodological gap in ROS biology and epilepsy research but also provides a versatile platform for studying redox regulation in neurological and inflammatory diseases and enabling mechanistic and therapeutic investigations.