Transcranial Theranostic Ultrasound for Pre-Planning and Blood-Brain Barrier Opening: A Feasibility Study Using an Imaging Phased Array In Vitro and In Vivo

Abstract

Focused ultrasound (FUS) for blood-brain barrier (BBB) opening is a safe, reversible and non-invasive strategy for targeted drug delivery to the brain, however extensive pre-planning strategies are necessary for successful FUS-mediated BBB opening through the structurally complex primate skull.

Objective: This study aims to demonstrate a pre-planning pipeline consisting of transcranial simulations and in vitro experimentation used to inform synchronous BBB opening and power cavitation imaging (PCI) with a single theranostic ultrasound (TUS) phased array.

Methods: Acoustic wave propagation simulation findings of pressure attenuation and focal shift through clinical-CT and micro-CT-based primate skull models were compared, while the latter were used to determine the impact of beam steering angle on focal shift and pressure attenuation. In vitro experimentation with a channel phantom enabled characterization of skull-induced receive focal shift (RFS), while in vivo BBB opening and PCI using in silico and in vitro pre-planning information was conducted using a custom Verasonics/MATLAB script.

Results: Simulations confirmed steering angle dependent transcranial focal shift and pressure attenuation, while in vitro experiments revealed minimal (0.30-1.50 mm) skull-induced RFS. In vivo rodent experiments with overlaid primate skull fragments demonstrated successful TUS-mediated BBB opening and spatially correlated power cavitation images (PCI) with regions of BBB opening on T₁-weighted magnetic resonance images (MRI).

Conclusion: We demonstrated the feasibility for TUS-mediated BBB opening in vivo using in silico and in vitro pre-planning information.

Significance: TUS as an ultrasound-guided modality for BBB opening could serve as a promising alternative to current FUS-mediated BBB opening configurations in the clinic.

Fig. 1.

Experimental setup for (a) transcranial pressure attenuation measurements, (b) in vitro PCI RFS calculations and (c) in vivo synchronous TUS-mediated BBB opening and PCI. Figure created using Biorender.

Fig. 2.

Simulated −6 dB focal regions through clinical CT vs micro-CT scans of an (a) NHP skull fragment and (b) a human skull fragment. White asterisks and cyan circles denote the 0 dB center of the focus simulated in free field, and through skulls, respectively. dB scale on color bar is self-normalized for each pressure plot. (c) A significant increase in pressure attenuation through an NHP skull fragment was observed in the simulation using a micro-CT compared to the clinical CT scan (****p<0.0001). (d) A significant increase in pressure attenuation through the human skull fragment was also observed using the micro-CT compared to the clinical CT scan (**p<0.01). (e) A significant decrease in axial focal shift was observed in the micro-CT simulation relative to the clinical CT simulation (**p<0.01) for the NHP skull, while (f) no statistically significant differences in focal shift were observed for the human skull. These results were determined by an unpaired Student’s t-test with n=6 lateral/elevational transducer positions. (g) Axial (top) and lateral (bottom) simulated focus profiles in free field (dashed line), through NHP skull, and human skull using micro-CT scans shown in (a-b). (h) Experimentally-determined axial (top) and lateral (bottom) focus profiles in free field, through NHP skull, and human skull.

Fig. 3.

Simulated impact of steering angle on pressure attenuation and focal shift. −6 dB focal regions of simulated focused transmits with varying steering angles through the (a) NHP skull and (b) human skull. Asterisk denotes center of the focus in free field, while circle denotes center of the focus after transcranial transmission. dB scale on color bar is self-normalized for each pressure plot. Steering angle dependent focal shift and attenuation through the NHP skull (c-d) and human skull fragment (e-f). Statistical significance determined by one-way ANOVA with post-hoc Tukey’s multiple comparisons test where, **p<0.01, ***p<0.001. ****p<0.0001, for n=6 simulation trials.

Fig. 4.

Transcranially-acquired PCI with overlaid B-mode images acquired of the channel phantom without skull interference for the (a) NHP skull, and (b) human skull, utilized to determine RFS. White arrows denote the center of the channel phantom on B-mode acquisitions.

Fig. 5.

In vivo BBB opening and PCI. Raw contrast enhanced T₁-weighted MRI (top panel) for (A) NHP skull and (B) human skull fragments. Thresholded regions of contrast enhancement used for ROC analysis with PCI (middle panel). Overlaid −6 dB focal regions of PCI (bottom panel) for (A) NHP skull and (B) human skull fragments. (C) Representative receiver operator characteristic (ROC) curves demonstrating correlation between contrast-enhanced regions of T₁-weighted MRI and −6 dB focal regions of PCI. (D) Area under the curve (AUC) for n=4 mice per group. Error bars denote the mean ± standard deviation.

Fig. 6.

Reversibility and safety of TUS-mediated BBB opening. Representative T₁-weighted MRI acquired 7 hours after TUS for (A) NHP skull and (B) human skull sonications depicting lack of contrast enhancement in regions of BBB opening (in Fig. 5A-B). Representative coronal H&E-stained brain slices of mouse brains sonicated bilaterally with an overlaid NHP skull fragment (C) or human skull fragment (D). No signs of damage were observed in any H&E-stained brain sections. Scale bars denote 1500 μm.