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Occlusion of the major cerebral artery usually results in brain hypoxic-ischemic injury, which evokes neuroinflammation and microglial activation. Activated microglia are considered a source of multiple neurotoxic factors, such as reactive oxygen species (ROS), in the central nervous system (CNS). We herein present a 3D-rendering brain imaging technique in an experimental rodent model of cerebral ischemia based on 2D magnetic images of superparamagnetic iron oxide nanoparticles (SPIONs) using the planar frequency mixing magnetic detection (p-FMMD) technique. A rat model of cerebral ischemia was established by unilateral middle cerebral artery occlusion with reperfusion (MCAO/R) injury. 2,3,5-Triphenyltetrazolium chloride (TTC) staining was performed to demonstrate the irreversibly damaged ischemic brain tissues, and double immunofluorescent labeling of OX6 (activated microglial marker) and ethidium (ROS marker) was conducted to confirm ROS generation in the activated microglia in the infarcted brain region. The ischemic brain sections treated with OX6-conjugated SPIONs were scanned using our p-FMMD system, yielding 2D images on the basis of the nonlinear magnetic characteristics inherent in SPIONs. The p-FMMD signal images representing microglia activation show an infarct ratio of 44.6 ± 7.1% compared to the contralateral counterpart, which is smaller than observed by TTC (60.9 ± 4.9%) or magnetic resonance imaging (MRI, 65.7 ± 2.7%). Furthermore, we developed a 3D-rendering brain imaging process based on the 2D p-FMMD signal images. The 3D reconstructed model showed a decreased ratio of coincidence of the ischemic regions compared with MRI models. In this study, we successfully conducted a feasibility test on whether our p-FMMD technology, a technique for signaling and imaging based on the nonlinearity of SPIONs, can be used to visualize the ischemic brain region in real time by detecting activated microglia in an MCAO/R animal model. Therefore, our method might allow for a different approach to analyze the pathophysiology of ischemic stroke through molecular imaging. Furthermore, we propose that this magnetic particle imaging (MPI) technique that detects the nonlinear magnetization properties of SPIONs could be applied not only to a stroke model but also to various types of pathophysiological studies as a new bioimaging tool.

Construction of 3D-rendering imaging of an ischemic rat brain model using the planar FMMD technique