Tracking ferroptosis in stroke models via an ESIPT-based near-infrared fluorescent probe for real-time monitoring of mitochondrial GSH dynamics.

Abstract

Ischemic stroke arises from vascular occlusion that triggers oxygen-glucose deprivation in neurons and glial cells, subsequently leading to metabolic collapse, excitotoxicity, and ultimately neuronal dysfunction and cell death. Ferroptosis, a form of regulated cell death characterized by iron accumulation and excess reactive oxygen species (ROS), is a key process in the pathological progression of ischemic stroke. Yet, current approaches for the investigation and treatment of ferroptosis in ischemia are limited by sensitivity of detection, spatial resolution to examine affected cells and tissues, and capacity for real-time monitoring. To address such challenges, we have developed a novel mitochondria-targeted fluorescent probe, XZTU-red-GSH, based on an excited-state intramolecular proton transfer (ESIPT) mechanism, such that the probe exhibits prominent, near-infrared emission at 720 nm and a large stokes shift of 315 nm, markedly enhancing fluorescence sensitivity and signal-to-noise ratio. Structurally, XZTU-red-GSH incorporates an N-methylpyridinium moiety for mitochondria targeting via electrostatic interactions, a 2,4-dinitrobenzenesulfonyl group as a selective response unit for glutathione (GSH), and a triethylene glycol monomethyl ether fragment to optimize the lipophilicity-hydrophilicity balance for optimizing blood-brain barrier permeability. In both oxygen-glucose deprivation/reperfusion (OGD/R) cellular models and middle cerebral artery occlusion (MCAO) animal models, the probe enabled in situ visualization of dynamic mitochondrial glutathione (mGSH) depletion during ferroptosis. Furthermore, our experiments with this probe demonstrated that the ferroptosis inhibitor Ferrostatin-1 (Fer-1) mitigates neuronal injury by preserving mGSH homeostasis. Taken together, our results indicate that XZTU-red-GSH is a novel tool to explore the dynamic features ferroptosis and ischemic stroke.