MitoGlow-ROS: A Novel Fluorescent Probe for Tracking Mitochondrial ROS in Cellular and Animal Models of Stroke and Neuroinflammation

Mitochondrial reactive oxygen species (mtROS) play a critical role in neuroinflammatory and neurodegenerative disorders, necessitating precise tools for their detection. This study validates MitoGlow-ROS as a highly sensitive and specific probe for quantifying mitochondrial superoxide in cell-free systems, PC12 cells, Lipopolysaccharide (LPS)-treated primary neuronal cultures, and in vivo LPS-treated Sprague-Dawley rats. In cell-free systems, MitoGlow-ROS exhibited a 45-fold dynamic range (R² = 0.97) and a limit of detection of 0.05 µM/min, outperforming MitoSOX Red, with enhanced reproducibility and specificity via its dynamic quenching mechanism. In PC12 cells with antimycin A, it detected a dose-dependent 5-fold ROS increase, reduced by 80% with N-acetylcysteine, confirming mitochondrial superoxide specificity. In LPS-treated neuronal cultures, MitoGlow-ROS captured a 3.4-fold ROS peak at 6 hours, correlating with robust early upregulation of inflammatory mediators (e.g., IL6: 1726-fold) and antioxidant enzymes (e.g., SOD2: 8.6-fold) at 3 hours. In vivo, it revealed a 4.1-fold hippocampal and 2.8-fold cortical ROS increase, validated by corresponding mRNA changes. Despite limitations in acute modeling and functional outcome assessments, MitoGlow-ROS’s sensitivity to subtle and significant mtROS changes positions it as a powerful tool for studying oxidative stress in stroke, Alzheimer’s disease, Parkinson’s disease, and other neuroinflammatory disorders, supporting future investigations into disease mechanisms and therapeutic interventions