Visualizing Peroxynitrite Fluxes in Endothelial Cells Reveals the Dynamic Progression of Brain Vascular Injury

Accumulating evidence suggests that formation of peroxynitrite (ONOO–) in the cerebral vasculature contributes to the progression of ischemic damage, while the underlying molecular mechanisms remain elusive. To fully understand ONOO– biology, efficient tools that can realize the real-time tracing of endogenous ONOO– fluxes are indispensable. While a few ONOO– fluorescent probes have been reported, direct visualization of ONOO– fluxes in the cerebral vasculature of live mice remains a challenge. Herein, we present a fluorescent switch-on probe (NP3) for ONOO– imaging. NP3 exhibits good specificity, fast response, and high sensitivity toward ONOO– both in vitro and in vivo. Moreover, NP3 is two-photon excitable and readily blood–brain barrier penetrable. These desired photophysical and pharmacokinetic properties endow NP3 with the capability to monitor brain vascular ONOO– generation after injury with excellent temporal and spatial resolution. As a proof of concept, NP3 has enabled the direct visualization of neurovascular ONOO– formation in ischemia progression in live mouse brain by use of two-photon laser scanning microscopy. Due to these favorable properties, NP3 holds great promise for visualizing endogenous peroxynitrite fluxes in a variety of pathophysiological progressions in vitro and in vivo.