Development of an In Vitro Model to Study Mechanisms of Ultrasound-Targeted Microbubble Cavitation-Mediated Blood-Brain Barrier Opening

Abstract

Objective: Ultrasound-targeted microbubble cavitation (UTMC)-mediated blood-brain barrier (BBB) opening is being explored as a method to increase drug delivery to the brain. This strategy has progressed to clinical trials for various neurological disorders, but the underlying cellular mechanisms are incompletely understood. In the study described here, a contact co-culture transwell model of the BBB was developed that can be used to determine the signaling cascade leading to increased BBB permeability.

Methods: This BBB model consists of bEnd.3 cells and C8-D1A astrocytes seeded on opposite sides of a transwell membrane. Pulsed ultrasound (US) is applied to lipid microbubbles (MBs), and the change in barrier permeability is measured via transendothelial electrical resistance and dextran flux. Live cell calcium imaging (Fluo-4 AM) is performed during UTMC treatment.

Results: This model exhibits important features of the BBB, including endothelial tight junctions, and is more restrictive than the endothelial cell (EC) monolayer alone. When US is applied to MBs in contact with the ECs, BBB permeability increases in this model by two mechanisms: UTMC induces pore formation in the EC membrane (sonoporation), leading to increased transcellular permeability, and UTMC causes formation of reversible inter-endothelial gaps, which increases paracellular permeability. Additionally, this study determines that calcium influx into ECs mediates the increase in BBB permeability after UTMC in this model.

Conclusion: Both transcellular and paracellular permeability can be used to increase drug delivery to the brain. Future studies can use this model to determine how UTMC-induced calcium-mediated signaling increases BBB permeability.

Keywords: Blood–brain barrier; Contrast agent; Microbubble; Sonoporation; Ultrasound.

Figure 1.. UTMC-mediated BBB opening was studied using an in vitro contact co-culture transwell model of the BBB.

(A) Schematic of the in vitro model of the BBB with ECs and astrocytes for permeability studies; Representative maximum intensity projections of confocal images of ECs in the co-culture labeled with (B) DAPI (blue) and anti-CD31 (red) antibody (20x); (C) anti-VE-cadherin (green) antibody (100x); (D) anti-ZO-1 (cyan) antibody (100x); and astrocytes co-labeled with DAPI (blue), phalloidin (red), and (E) anti-GFAP (green) antibody (60x); (F) anti-AQP4 (green) antibody (60x). BBB = blood-brain barrier; EC = endothelial cell; UTMC = ultrasound-targeted microbubble cavitation.

Figure 2.. The co-culture had stronger barrier function than the monolayer alone.

The co-culture (EC + Astrocytes) had (A) higher TEER and (B) less 10 kDa Texas Red dextran flux than the monolayer (EC only). Data represented as mean ± SD (n=3-12). For two groups, two-tailed student’s t-tests were run, and for multiple comparisons (*p<0.05). EC = endothelial cell; TEER = transendothelial electrical resistance.

Figure 3.. UTMC increased sonoporation without decreasing cell viability or cell coverage.

After UTMC, there was no significant change in (A) cell viability (PI+/Calcein-) nor (B) percentage of transwell area covered by ECs; and (C) an increase in the percentage of PI+/Calcein+ cells. Data represented as mean ± SD (n=3). Two-tailed student’s t-tests were run (*p<0.05). ns = not significant. EC = endothelial cell; PI = propidium iodide; UTMC = ultrasound-targeted microbubble cavitation.

Figure 4.. UTMC increased endothelial barrier permeability.

After UTMC, there was: (A) a decrease in TEER; (B) an increase in 10 kDa Texas Red dextran flux for at least one hour. Data represented as mean ± SD (n=3-5). For two groups, two-tailed student’s t-tests were run, and for multiple comparisons, one-way ANOVA analyses with correction for post-hoc t-tests were run (*p<0.05). TEER = transendothelial electrical resistance; UTMC = ultrasound-targeted microbubble cavitation.

Figure 5.. UTMC increased paracellular gaps.

Representative maximum intensity projections of confocal images of ECs after UTMC stained with anti-CD31 (cyan) antibody, co-labeled with DAPI (blue) and paracellular gaps circled in red at (A) 0 kPa (B) 250 kPa (60x). After UTMC, there was an increase in the (C) number of interendothelial gaps and (D) total gap area per field (60x). Data represented as mean ± SD (n=3). One-way ANOVA analyses with correction for post-hoc t-tests were run (*p<0.05). EC = endothelial cell; UTMC = ultrasound-targeted microbubble cavitation.

Figure 6.. UTMC-mediated BBB opening was calcium dependent.

(A) Schematic of the in vitro model of the BBB with ECs and astrocytes for live cell imaging; (B) After UTMC, cytosolic calcium increased in ECs (percentage of Fluo-4 AM+ cells; also shown in Supplementary Video 3A and 3B). This was decreased when cells were in calcium-free media. (C) The wave of calcium influx beginning at a sonoporated EC (-•-) propagated into cells within 50 μm of the index cell over time. (D) The decrease in TEER after UTMC was attenuated when cells were incubated in calcium-free media. Data represented as mean ± SD (n=3). Two-tailed student’s t-tests were run (*p<0.05). BBB = blood-brain barrier; EC = endothelial cell; TEER = transendothelial electrical resistance; UTMC = ultrasound-targeted microbubble cavitation.