Evaluating the Protective Effects of Mitochondrial Glutathione on Cerebral Ischemia/Reperfusion Injury via Near-Infrared Fluorescence Imaging.

Cerebral ischemia/reperfusion (I/R) is common and intractable in the clinic, associated with the outburst of reactive oxygen species (ROS) in mitochondria. Although numerous research studies have been conducted to prove the protective-effect roles of glutathione (GSH) in this event, the changes in GSH concentrations in living cells remain largely unexplained, and there is scarce evidence by fluorescence imaging for its roles. Herein we have designed and synthesized two distinctive "off-on" near-infrared (NIR) fluorescent probes BCy-SeSe and BCy-SS based on a new fluorophore BCy-Keto for specific response to mitochondrial GSH changes during the cerebral I/R process. Both of them exhibit powerful targeting capability in mitochondria and excellent photophysical properties toward endogenous GSH with high selectivity and sensitivity. In contrast to BCy-SS, BCy-SeSe was screened for biological application on account of its faster response rate. We have utilized BCy-SeSe to real-time image GSH during the cerebral I/R process in living cells and the mice focal cerebral ischemia model (middle cerebral artery occlusion, MCAO), and these intensive studies revealed that low GSH levels were associated with aggravation of apoptosis and cerebral infarction. Pretreatment with GSH synthase inhibitor aggravates damage while GSH-ester alleviates damage, confirming that GSH is effective on the cerebrum for protection from I/R. All the results demonstrated that the probes were powerful tools for investigating mitochondrial GSH during the I/R process in living cells and in vivo.