To assess BBB closure mice received intraperitoneal injections of gadolinium contrast agent, Omniscan (0.5 ml/kg), which circulated 20 minutes prior to image acquisition. For the purpose of this study longitudinal intravenous injections in the mouse tail vein were a technical limitation. A catheter cannot be left in for extended period of time, nor could the tail vein be repeatedly injected within the short time frame. Upon inducing BBB permeability, the initial enhancement and coordinates of each focal spot were recorded. In subsequent follow-up images the same coordinates were used to quantify enhancement (Fig. (Fig.1C);1C); BBB closure was defined when there was less than two standard deviations of the enhanced relative to the unenhanced hemisphere. Each focal spot represents a distinct vasculature composition and a unique acoustic emission, as detected by the feedback controller. As such, each focal spot was considered independently. Gadolinium contrast enhancement was quantified in MATLAB (Mathworks, Natick, MA) by finding the mean pixel intensity of a 3×3 voxel region of interest surrounding the point of maximum enhancement.

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