In vivo electron paramagnetic resonance spectroscopy and imaging of mouse brains using an acetoxymethyl ester nitroxide probe

Nitroxide probes have recently been used as contrast agents in magnetic resonance-based imaging modalities to monitor physiological parameters such as redox status and pH. In particular, lipophilic nitroxides are used as contrast agents, specifically for mouse brain imaging. However, their relatively short contrast enhancement duration and limited brain distribution have prompted strong interest in alternative nitroxide probes. Owing to its blood–brain barrier (BBB) permeability and long retention within cells, the acetoxymethyl ester nitroxide acetoxymethyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (AMCP) was synthesized. Subsequently, the dynamic distribution of AMCP in the mouse head was examined using our recent electron paramagnetic resonance (EPR) imager with fast magnetic field scanning capability for rapid data acquisition. In vivo EPR spectra of mouse heads were acquired under a 50 ms field scan over 6 mT at 9 s intervals, revealing the sequential process of AMCP transport across the BBB, enzymatic hydrolysis by esterase to 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidine-1-oxyl, and distribution within the brain. The distribution and retention of AMCP in the mouse brain were examined using a three-dimensional EPR imager and magnetic resonance imaging. These results were compared with those obtained using a similar pyrrolidine nitroxide compound, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl, which contains an ester group that is minimally hydrolyzed by esterase in vivo. Overall, our findings indicate that, when combined with our EPR imager, AMCP could be a powerful contrast agent and useful for monitoring and evaluating physiological parameters in the brain.