Reversible and localized blood-brain barrier disruption (BBBD) using focused ultrasound (FUS) in combination with intravascularly administered microbubbles (MBs) has been established as a non-invasive method for drug delivery to the brain. Using two-photon fluorescence microscopy (2PFM), we imaged the cerebral vasculature during BBBD and observed the extravasation of fluorescent dye in real-time in vivo. We measured the enhanced permeability upon BBBD for both 10kDa and 70kDa dextran conjugated Texas Red (TR) at the acoustic pressure range of 0.2-0.8 MPa and found permeability constants of TR10kDa and TR70kDa vary from 0.0006 to 0.0359 min−1 and 0.0003 to 0.0231 min−1, respectively. For both substances, a linear regression was applied on the permeability constant against the acoustic pressure and the slope from best-fit was found to be 0.039±0.005 min−1/MPa and 0.018±0.005 min−1/MPa, respectively. In addition, the pressure threshold for successfully induced BBBD was confirmed to be 0.4-0.6 MPa. Finally, we identified two types of leakage kinetics (fast and slow) that exhibit distinct permeability constants and temporal disruption onsets, as well as demonstrated their correlations with the applied acoustic pressure and vessel diameter. Direct assessment of vascular permeability and insights on its dependency on acoustic pressure, vessel size and leakage kinetics are important for treatment strategies of BBBD-based drug delivery.
Blood-brain barrier (BBB) disruption can be achieved with ultrasound (US) and circulating microbubble (MB) contrast agent. Using dorsal US sonication and Definity, an MB contrast agent, responses of the cortical cerebral vasculature to BBB opening were observed with varying acoustic peak negative pressure (0.071 to 0.25 MPa) under two-photon microscope. Wistar rats with a craniotomy were sonicated with a single piezoelectric transducer following the intravenous injection of Texas Red for visualization of vasculature and leakage from BBB opening. Based on timedependent intensity change in the extravascular area, the leakage was classified into three types: fast, sustained, and slow. Fast leakage was characterized by a rapid increase to peak intensity during sonication, but a decrease afterwards, occurring at all pressures and vessels sizes analyzed in our study. Sustained leakage was indicated by a similar, immediate increase to peak intensity but one that remained elevated for the duration of imaging, occurring at low-to-intermediate pressures. Slow leakage began 5 to 15 minutes after sonication, dominating at low pressures, and was more prevalent among smaller vessels than fast and sustained leakage. Our study showed the possibility of controlling leakage type and vessel size in US-induced BBB opening through varying acoustic pressure.
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