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-[(11)C]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-[(11)C]methyl-hydroxyfasudil were investigated in the brains of mice.

A 90-min dynamic scan was performed following intravenous infusion of N-[(11)C]methyl-hydroxyfasudil.

The uptake of N-[(11)C]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-[(11)C]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-[(11)C]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-[(11)C]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-[(11)C]methyl-hydroxyfasudil binds to the active form of Rho-kinase. PET imaging using N-[(11)C]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.