Nitroxides are stable free radicals that have unique antioxidant properties. Because of their ability to interact with free radicals, they have been used for many years as biophysical tools [1]. Moreover, during the past two decades, numerous interesting biochemical properties have been discovered in nitroxides that have been used for many biomedical applications. For instance, piperidine nitroxide (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl: Tempol) is presently being tested in clinical trials. However, although these piperidine nitroxides have been extensively studied for therapeutic applications, details concerning the distribution and kinetics of Tempol in vivo have not been thoroughly studied. In particular, it is not clear whether Tempol enters the brain as a paramagnetic material containing an unpaired electron. To resolve this matter, an electron paramagnetic resonance (EPR) imaging study of mouse brains was conducted by using a modified EPR imager with a piperidine nitroxide imaging probe. Three-dimensional (3D) EPR images of mouse brains could be taken in less than 10 s by the modified EPR imager, therefore making it possible to visualize the exact distribution of Tempol in the brain. The obtained EPR images clearly showed that Tempol with an unpaired electron could enter the brain. EPR images also visualized the effect of antioxidants present in mouse brains on the lifetime of piperidine nitroxide.

Control of magnetic field scanning and magnetic field gradient: To reduce the total acquisition time, analog signals were used to drive the Helmholtz coil pair for field scanning and field gradient coils. These signals were generated by a Field Programmable Gate Array (FPGA) developing board (DE0-nano), and were fed to the bipolar power supplies for field scanning and field gradient. For 3D imaging, the control sequences for four different projections (81, 126, 181, 246) and six different durations of field scanning (54~132 ms) were installed in the FPGA board. Animals: Male C57BL/6 mice aged 5 to 7 weeks with body weights of 20–25 g were used. Paramagnetic nitroxides: Piperidine nitroxides, Tempol and 4-oxo-2,2,6,6-tetrametylpiperidine-1-oxyl (Tempone) (Figure 1), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nitroxide solutions were injected by tail vein cannulation under isoflurane anesthesia. MRI measurements: MRI of mouse heads was acquired using an MRmini scanner (MR Technology, Tsukuba, Japan) with a 0.5-T permanent magnet. EPR imaging measurements: All EPR images were acquired using an in-house built 750-MHz continuous wave EPR imaging system.For 3D EPR images of the living mouse heads obtained by the modified EPR imager, the total imaging times for 121 and 181 projections were 6.3 and 9.1 s, respectively. Immediately after injection of piperidine nitroxide, Tempol or Tempone, through the tail vein, a 3D EPR data set was obtained every 10 s. To show the detailed distribution of Tempol and Tempone in mouse heads, 2D EPR images were obtained from 3D EPR imaging data. MRI of mouse heads was taken in advance for co-registration, on which the EPR image was overlaid. 2D EPR images obtained 10 s after injection of nitroxides and co-registered images for both Tempol and Tempone are shown in Figure 2, respectively. Many previous in vitro studies have showed that Tempol and Tempone enter the brain through the blood brain barrier (BBB), but to date there have been no direct evidence indicating that Tempol and Tempone enter the brain as being paramagnetic materials, i.e., with an unpaired electron. However, the co-registered images in Figure 2 clearly show Tempol and Tempone in the brain with an unpaired electron. To estimate the lifetime of nitroxides that entered the brain, the pixel-based reduction rate constant of nitroxides was calculated from a series of temporal 3D EPR images. The obtained rate constants were displayed as a 2D map for both Tempol and Tempone (Figure 3). These maps also showed that Tempol and Tempone could exist within the brain with an unpaired electron. Both nitroxides had a lifetime of about 40 s in the brain. Using this methodology, we also visualized the effect of a BBB-permeable anti-oxidant, glutathione monoethyl ester, on the lifetime of nitroxides in the brain. The EPR imaging method is a possible means to follow the change in the concentration of antioxidants in the brain.Through a modified EPR imaging system, we successfully found that the BBB permeable piperidine nitroxides, Tempol and Tempone, entered the mouse brains with an unpaired electron. This method allows us to study the effect of antioxidants on the redox status in the brain under oxidative stress.