Evaluation of Rho-Kinase Activity in Mice Brain Using N-[11C]Methyl-hydroxyfasudil with Positron Emission Tomography

Rho is a small molecular weight GTP-binding protein and works as a molecular shuttling switch between an active (GTP-bound) and inactive (GDP-bound) state. Rho is known to be involved in cell motility, cell adhesion, and cytokinesis through actin cytoskeleton reorganization. The GTP-bound form of Rho interacts with its specific downstream target, triggering intracellular signaling cascades. Rho effectors such as Rho-kinases have been isolated on the basis of their selective binding to the GTP-bound form of Rho. Rho-kinase is thought to have an important role in the pathogenesis of a variety of neurological diseases because activation of the Rho/Rho-kinase pathway has been observed in various central nervous system disorders. Previous histochemical studies have shown multiple molecular mechanisms for the regulation of Rho-kinase. Neuroimaging of Rho/Rho-kinase has rarely been studied because of a lack of appropriate radiotracers. Recently, N-[C-11]methyl-hydroxyfasudil, a new radiotracer for positron emission tomography (PET), has been introduced to measure Rho-kinase activity. In this study, the regional distribution and kinetics of N-[C-11]methyl-hydroxyfasudil were investigated in the brains of mice. A 90-min dynamic scan was performed following intravenous infusion of N-[C-11]methyl-hydroxyfasudil. The uptake of N-[C-11]methyl-hydroxyfasudil reached a maximum within 5 min and gradually decreased in all organs. The standard uptake values (SUVs) in the brain, liver, and kidney on average between 30 to 60 min were 0.17 +/- 0.03, 0.76 +/- 0.18, and 0.62 +/- 0.18 and from 60 to 90 min were 0.15 +/- 0.01, 0.69 +/- 0.33, and 0.64 +/- 0.17, respectively. N-[C-11]Methyl-hydroxyfasudil showed a widespread distribution throughout the brain, with low levels of radioactivity. Radioactivity concentration in plasma at 90 min after injection of N-[C-11]methyl-hydroxyfasudil resulted in SUVs in the control and fasudil pretreatment of 0.0013 and 0.0023 +/- 0.0008, respectively. Compared to normal control mice, about twofold higher radioactivity concentration was observed in fasudil-pretreated mice. In a cold brain injury mouse model, accumulation of N-[C-11]methyl-hydroxyfasudil was slightly higher at the injury site than that at the control site, and the difference was statistically significant in the 24 h after injury group (P < 0.05). These results suggest that following brain injury, N-[C-11]methyl-hydroxyfasudil binds to the active form of Rho-kinase. PET imaging using N-[C-11]methyl-hydroxyfasudil could provide new insights into the pathophysiology of a variety of neurological disorders including stroke, inflammatory diseases, demyelinating diseases, Alzheimer's disease, and neuropathic pain.