Drug delivery to the brain by focused ultrasound induced blood–brain barrier disruption: Quantitative evaluation of enhanced permeability of cerebral vasculature using two-photon microscopy

Abstract

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.

Disrupting the blood–brain barrier by focused ultrasound induces sterile inflammation

“…pFUS+MB has been reported to cause hemodynamic alterations in the brain associated with temporary vasoconstriction, as seen with BBBD (31,32). The molecular effects on the NVU associated with BBBD following pFUS+MB have received limited attention.…”

Quantification of transient increase of the blood–brain barrier permeability to macromolecules by optimized focused ultrasound combined with microbubbles

“…6,7,11,15,21,25 Based on prior literature, 6,7,10,21 we fixed the MB size to ~2 μm, MB concentration to ~1.7×10 6 bubbles/mL, and sonication time to ~1 minute. Subsequently, we adjusted the voltage and BRR while keeping the duty cycle constant at 5% to generate different acoustic powers.…”

“…Nhan et al 25 used two-photon microscopy to investigate the reversible and localized BBB disruption induced by FUS in the presence of MBs by imaging the cerebral vasculature during BBB disruption and observing the extravasation of fluorescently labeled solutes in real-time in vivo. They found that the threshold acoustic pressure for a successful induction in BBB disruption was 0.4-0.6 MPa by combining the MBs with a similar size and a reduced concentration as those used in the current study.…”

“…23,24 To overcome aforementioned limitations, two-photon microscopy can be employed to quantitatively access the enhanced permeability of the cerebral vasculature after FUS. 25 In addition to micrometer spatial resolution, two-photon microscopy offers the advantage of deep tissue penetration, which is essential for the BBB permeability measurement. However, in the studies from Nhan et al 25 the MBs for enhancing the FUS effect and the fluorescently labeled test solutes were injected via the animal tail vein.…”

“…25 In addition to micrometer spatial resolution, two-photon microscopy offers the advantage of deep tissue penetration, which is essential for the BBB permeability measurement. However, in the studies from Nhan et al 25 the MBs for enhancing the FUS effect and the fluorescently labeled test solutes were injected via the animal tail vein. This administration route leads to the uneven distribution of MBs and uncertainty in the MB concentration in both the FUS-stimulated region and in the test solute concentration in cerebral microvessels, resulting in inaccurate assessments of the dynamics of FUSenhanced BBB permeability.…”