Diameter-dependent assessment of microvascular leakage following ultrasound-mediated blood-brain barrier opening

Blood brain barrier disruption (BBBD) using focused ultrasound (FUS) and microbubbles (MB) is an effective tool for therapeutic delivery to the brain. BBBD depends to a great extent on MB oscillations. Because the brain vasculature is heterogenic in diameter, reduced MB oscillations in smaller blood vessels, together with a lower number of MBs in capillaries, can lead to variations in BBBD. Therefore, evaluating the impact of microvasculature diameter on BBBD is of great importance. We present a method to characterize molecules extravasation following FUS-mediated BBBD, at a single blood vessel resolution. Evans blue (EB) leakage was used as marker for BBBD, whereas blood vessels localization was done using FITC labeled Dextran. Automated image processing pipeline was developed to quantify the extent of extravasation as function of microvasculature diameter, including a wide range of vascular morphological parameters. Variations in MB vibrational response were observed in blood vessel mimicking fibers with varied diameters. Higher peak negative pressures (PNP) were required to initiate stable cavitation in fibers with smaller diameters. In vivo in the treated brains, EB extravasation increased as a function of blood vessel diameter. The percentage of strong BBBD blood vessels increased from 9.75% for 2-3 μm blood vessels to 91.67% for 9-10 μm. Using this method, it is possible to conduct a diameter-dependent analysis that measures vascular leakage resulting from FUS-mediated BBBD at a single blood vessel resolution.