Animal preparation
All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Washington University in St. Louis in accordance with the National Institutes of Health Guidelines for animal research. This study is reported in accordance with ARRIVE guidelines. A total of 15 mice (Swiss, 6–8 weeks old, female, Charles River Laboratory, Wilmington, MA) were used. These mice were randomly divided into three groups to compare the outcome of FUS-BBBO under the three coupling conditions: “oil + hairs” (n = 5), “ultrasound gel + no hair” (n = 5), and “ultrasound gel + hairs” (n = 5).
FUS-BBBO experimental setup
An MR-guided FUS system (Image Guided Therapy, Pessac, France) was used to perform the FUS-BBBO procedure in mice. A schematic diagram of the experimental system is shown in Fig. 1A. This system was used in our previous MR-guided FUS-BBBO studies10,20. The system consisted of an MRI-compatible FUS transducer (Imasonic, Voray-sur-l’Ognon, France) made of a seven-element annular array with a center frequency of 1.5 MHz, an aperture of 25 mm, and a radius of curvature of 20 mm. The system was integrated into a 9.4 T small animal MRI scanner (Bruker, Billerica, MA, USA). The transducer was connected to an MRI-compatible piezoelectric motor, allowing the position of the transducer to be mechanically adjusted in the lateral directions (along the x- and y-axes, Fig. 1A). The axial and lateral full widths at half maximums of the FUS transducer were 5.5 mm and 1.2 mm, respectively. The acoustic pressure reported in this study was corrected for 18% mouse skull insertion loss10,20. A passive cavitation detection (PCD) sensor integrated at the center of the FUS transducer had a center frequency of 1.6 MHz and a − 6-dB bandwidth of 754 kHz. The signals detected with the PCD sensor were acquired via the PicoScope (5244B, Pico Technology, Cambridgeshire, UK) to monitor cavitation events. The transducer set (FUS transducer and PCD) was connected to a water balloon filled with deionized and degassed water.
Acoustic coupling methods
The water balloon of the transducer was coupled to the mouse head using different coupling methods, as shown in Fig. 1A and B.
For the “oil + hairs” group, mineral oil (Walgreen Company, Deerfield, IL) was first poured into a weighing paper bowl and waited for at least 5 min to allow trapped bubbles, if there were any, to float and dissipate from the oil. A portion of the mineral oil was then loaded in a syringe. The rest was used to soak cotton swaps. Drops of oil were then applied onto the mouse’s head using a syringe, and the oil was gently spread and brushed with oil-dipped cotton swaps. Then, 1–2 mL of oil was added on top of the hairs using a syringe. The height of the FUS transducer was then adjusted such that the transducer membrane was in contact with the oil.
For the “ultrasound gel + no hair” group, hairs were removed using Nair (Church & Dwight Co., Princeton, NJ, USA), and the scalp was thoroughly cleaned using alcohol pads. Afterward, degassed ultrasound gel (Aquasonic, Parker Laboratories, Inc., Fairfield, NJ, USA) was applied, and the FUS transducer was positioned to couple with the ultrasound gel.
For the “ultrasound gel + hairs” group, hairs were first thoroughly cleaned using alcohol pads. Degassed water was added to the hairs, and ultrasound gel was applied to the wetted hairs. The FUS transducer was then coupled with the ultrasound gel.
FUS-BBBO procedure
Following the completion of acoustic coupling, T2-weighted MRI scans (TR/TE: 2200/35; slice thickness: 0.5 mm; in-plane resolution: 0.125 mm; matrix size: 256 × 256) were performed to acquire the relative location of the FUS transducer to the brain. The left striatum was chosen as the targeted brain location (FUS +), and the contralateral side was chosen as the nonsonicated control (FUS-). The transducer set (FUS transducer and PCD) was turned on, and during sonication by each FUS pulse, acoustic emission from microbubbles was recorded by the PCD. The acoustic parameters were kept the same among all three groups (0.6 MPa peak negative pressure in situ, 5 Hz pulse repetition frequency, 10,000 cycles, 3.3% duty cycle, and 3 min sonication duration). Thirty seconds after the onset of FUS sonication, commercial microbubbles (Definity, Lantheus Medical Imaging, North Billerica, MA, USA) were administered intravenously at a concentration of \(8\times {10}^{8}\) bubbles/mL and a total volume of 30 µL, followed by a saline flush. Immediately after FUS treatment, 4% Evans blue was delivered intravenously as a model drug.
PCD signal processing
Similar to our past publication10, a custom MATLAB script was written to process the acquired PCD data for the evaluation of stable cavitation doses. Briefly, baseline PCD data was acquired during the initial 30 s sonication before microbubble injection. After microbubbles were injected, PCD data were acquired until the sonication ended. The stable cavitation dose was calculated with the following steps: (1) The mean of the pre-microbubble injection stable cavitation (SC) level was calculated by averaging the SC levels observed before microbubble injection. The SC level was assessed in the frequency domain by summing the amplitude at the second harmonic (3.0 MHz) within a ± 0.02 MHz bandwidth. (2) The post-microbubble injection SC level was determined by subtracting the mean of the pre-microbubble SC level from the SC level calculated at each time point. (3) The stable cavitation dose was then quantified by summing the SC levels between the time after microbubble injection and the end of sonication. These steps ensured the consideration of the variations in the baseline SC level for individual mice.
MRI evaluation of the acoustic coupling quality
T2-weighted MRI scan (TR/TE, 4228/35; slice thickness: 0.5 mm; in-plane resolution: 0.125 mm; matrix size: 256 × 256) was performed at the top of the mouse head to evaluate the quality of the acoustic coupling. As a quantitative assessment, we calculated the number of air bubbles trapped in the coupling medium within a region of interest (ROI) at the interface between the coupling medium and the hair/skin of the mice. The size of the ROI (40 × 24 pixels, pixel width of 0.125 mm) was kept consistent throughout all objects. The location of ROI was fixed on the posterior portion of the skin/hair-coupling medium interface region, which appeared as the gray area in the T2-weighted MRI images. The air bubbles inside the ROI were then extracted by post-processing using ImageJ21.
FUS-BBBO outcome assessment
The FUS-BBBO outcome was assessed in vivo using contrast-enhanced T1-weighted MRI and ex vivo by fluorescence imaging of brain slices.
In vivo, contrast-enhanced T1-weighted MRI scan (TR/TE: 20/5; slice thickness: 0.13 mm; in-plane resolution: 0.13 mm; matrix size: 120 × 240; flip angle: 20°) was performed to evaluate the outcome of FUS-BBBO based on the extravasation of the MRI contrast agent gadobenate dimeglumin (Gd-BOPTA; MultiHance, Bracco Diagnostics Inc., Monrow Township, NJ) from the blood circulation into brain tissue. BBBO volume was calculated by comparing the contrast-enhanced volume in the T1-weighted images on the FUS + and FUS- sides using a custom MATLAB script reported in our previous publications10,20,22. Briefly, the contrast-enhanced volume for each mouse was calculated by the sum of voxels in the FUS + side with an intensity above the mean plus three times the standard deviation of the FUS- side for each individual scan slice.
All mice were sacrificed under vaporized isoflurane anesthesia at around 30 min after FUS sonication by transcardial perfusion with 30 mL 1 × PBS for 5 min. Brains were harvested and fixed in 4% paraformaldehyde for at least 24 h. The brains were then transversely cut into 1 mm slices using a brain matrix (RBM-4000C, ASI Instruments Inc., MI, USA) and imaged using the Pearl Trilogy Image System (LI-COR, Lincoln, NE, USA). Evans blue delivery outcome was quantified using the system’s built-in software (Image Studio Lite, LI-COR, Lincoln, NE, USA). The fluorescence intensity within the FUS + side was summed and normalized by the sum of the fluorescence intensity of the contralateral side of the striatum for each individual brain slice.
Statistical analysis
Statistical analyses were performed using GraphPad Prism (Version 9.0, La Jolla, CA, USA). Differences among multiple groups were determined using ordinary one-way ANOVA with group-wise comparisons. A p value < 0.05 was used to determine statistical significance.