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.