Author(s): Pennapa Chonpathompikunlert 1 , Ching-Hsiang Fan 2 , Yuki Ozaki 1 , Toru Yoshitomi 1 , Chih-Kuang Yeh 2 , Yukio Nagasaki [*] 3
brain edema; contrast-enhanced ultrasound; focused ultrasound; intracerebral hemorrhage; microbubbles; nitroxide-radical containing nanoparticle; oxidative stress; reactive oxygen species; redox-polymer drug; self-assembling polymer micelle
Local blood-brain barrier (BBB) opening is an advantageous approach to deliver active substances and drugs to the brain  . However, sonication used for focal BBB opening may result in erythrocyte extravasation and microhemorrhage capillary rupture, as evidenced by light microscopy  , multiphoton imaging  and scanning electron microscopy for detection of endothelial damage  . It is, thus, strongly reliable that physical damage to endothelial cells may play a role in sonication-induced changes of BBB permeability [5,6] . Contrast-enhanced T1 -weighted imaging can detect an increase in BBB permeability, but fails to detect possible hemorrhage caused by the focused ultrasound exposure, which may prevent the development of its clinical applications. Moreover, Liu et al. used magnetic resonance susceptibility-weighted imaging to identify tissue hemorrhage associated with disruption of the BBB induced by focused ultrasound in a rat model  . They also mentioned that focused ultrasound with microbubbles may provide a reliable experimental model to investigate cerebral hemorrhage, with inherent advantages over other methods such as intracerebral injection of blood or collagenase. In this study, we tried to develop a hemorrhage model using clinical cerebral hemorrhage symptoms such as neurological deficit and brain edema to detect hemorrhage induced by focused ultrasound sonication (FUS) coupled with microbubbles, with inherent advantages compared with other methods such as intracerebral injection of blood [8,9] or collagenase [10,11] .
Clinical and animal studies have provided evidence that inflammation and oxidative stress from reactive oxygen species (ROS) are involved in secondary brain injury after intracerebral hemorrhage (ICH) [12-14] . ROS are usually scavenged by antioxidant enzymes, primarily superoxide dismutase (SOD), by catalyzing the dismutation reaction of the superoxide anion to hydrogen peroxide. Catalase and glutathione peroxidase, on the other hand, protect cells from the toxic effects of hydrogen peroxide by catalyzing its decomposition into water  . Under normal conditions, there is a balance between free radical generation and free radical scavenging; however, under hemorrhage conditions, the imbalance between the two processes results in oxidative stress and contributes to several pathophysiological conditions.
The strategy of exogenous delivery of the native form of SOD to neutralize the deleterious effect of ROS has been ineffective because SOD has a short half-life ([proportional to]6 min) in circulation and poor permeability across the BBB  . Alternatively, antioxidant drugs, such as nitroxide radical compounds, have been employed for suppression of such oxidative compounds. The nitroxide radical compound 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) is one of the strongest antioxidants that catalytically scavenges ROS [17,18] . Under in vivo conditions, however, these low molecular weight nitroxide compounds have several problems, including nonspecific dispersion in normal tissues, preferential renal clearance and rapid reduction to the corresponding hydroxylamine form. To solve these issues, we have designed and developed redox polymer self-assembled nanoparticles (nitroxide radical-containing nanoparticles [RNPs]), approximately 40 nm in diameter. Since the nitroxide...