FUS-BBBD treatment

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. C57BL/6 female mice (6–8 weeks, 20–25 g body weight, n = 18 in total) were purchased from Charles River Laboratory (Wilmington, MA, USA). Following a procedure described in our previous publication20, the animals were prepared for FUS sonication by removing the fur on the mouse head and coupled to a water container using ultrasound gel.

The prepared mice were sonicated using a commercially available ultrasound image-guided FUS system (VIFU 2000, Alpinion, Bothell, WA, USA) that integrated a FUS system with a software programmable ultrasound imaging system. This system used a FUS transducer with a center frequency of 1.5 MHz, a focal depth of 60 mm, an aperture of 60 mm, and a circular central opening of 38 mm. A linear array (L8–17, Alpinion, Seoul, Korea) with a bandwidth of 8–17 MHz and a center frequency of 12 MHz was inserted into the FUS transducer center opening. The ultrasound imaging plane was aligned with the FUS focal plane.

The ultrasound imaging system was used for both treatment planning and PCI. Treatment planning was performed with the assistant of a metal grid using a previously published method to align the FUS transducer focus at the left brainstem20. Size-isolated microbubbles with a median diameter of 4–5 μm were prepared in-house according to a previously described protocol45 and diluted using sterile saline to a final concentration of about 8 × 108 number of microbubbles per ml. The diluted microbubbles (volume = 25 μL) were administered by a bolus injection via the tail vein. Immediately after injection (~9 s), the mice were treated by the FUS system using the following parameters: center frequency = 1.5 MHz, peak negative pressure = 0.61 MPa, pulse length = 6.7 ms, pulse repetition frequency = 5 Hz, and sonication duration = 120 s. After treatment, mice were put back into their cages for them to recover from anesthesia.

The pressure amplitude and beam dimensions of the FUS transducer were calibrated using a needle hydrophone (HGL-0200, Onda, CA, USA) in a degassed water tank before the experiment. The pressures reported here were the measured hydrophone peak negative pressures corrected for 18% mouse skull attenuation3. The full width at half maximum (FWHM) dimensions of the axial and lateral beams were 6.04 mm and 0.62 mm, respectively.

PCI data acquisition and processing

The ultrasound imaging system was programmed to first operate in the pulse-echo mode to acquire a B-mode image for identifying the location of the mouse skull during PCI post-processing. The system was then operated at the passive mode for acquiring two sets of PCI data. The first PCI data acquisition was performed with FUS sonication on but before the injection of microbubbles to define the background cavitation noise level. The second PCI data acquisition was performed immediately after microbubble injection and in synchronized with the FUS sonication. During the sonication of each FUS pulse, one PCI frame was acquired with an acquisition duration of 400 μs.

In reference to a PCI method used by Haworth et al.35, a frequency-domain PCI algorithm was written in Matlab (Mathworks inc., Natick, MA, USA) to proccess the acquired PCI data offline through the following procedure: (1) Apply Butterworth bandpass filtering around the imaging probe bandwidth of 8–17 MHz to reduce contributions from FUS source signals; (2) Apply a phase shift to the signal acquired by each element in the frequency domain (equivalent to a time delay in temporal domain) based on the propagation times between the receiving element and the spatial location the pixel represents; (3) Sum the phase-shifted waveforms across all the elements for each pixel and its energy is computed by calculating the square of the summed waveforms; Parallel computation using Graphics Processing Units (GPUs) was integrated into the Matlab code to accelerate the calculation speed; (4) Calculate the mean amplitude of the spectrum within selected harmonic bandwidths (0.3 MHz window around all harmonics and superharmonics) for each pixel to generate SC maps; the mean amplitudes of braodband signal falling within frequency bands between the harmonic and ultraharmonic bands were used to calculate the IC levels and generate the IC maps; (5) The SC and IC maps obtained from each FUS treatment were integrated over time to obtain the SCD and ICD maps, respectively.

In vivo MicroPET/CT image acquisition and processing

The mice were imaged by the Inveon PET/CT system (Siemens, Knoxville, TN) at 24 h after intravenous injection of 64Cu-AuNCs at a concentration ~10 MBq following the FUS-treatment. Details on the 64Cu-AuNCs manufacture and characterization were reported in our previous publications49. The 64Cu-AuNCs had a homogeneous size distribution with the hydrodynamic diameter = 5.60 ± 0.50 nm and zeta potential = −0.40 ± 0.11 mV. In our previous study, we performed PET imaging at 1 h, 4 h, and 24 h post treatment and found the 64Cu-AuNCs were almost cleared from the blood circulation at the 24 h time point20. Therefore, in the current study, we chose to perform the microPET/CT scans at the 24 h time point as it allowed us to more accurately quantify the 64Cu-AuNCs concentrations in the brain tissue compared with earlier time points by eliminating the interference of the 64Cu-AuNCs in the blood.

The acquired PET images were corrected for attenuation, scatter, normalization, and camera dead time and co-registered with CT images. The PET images were reconstructed with the maximum a posteriori (MAP) algorithm. Images were analyzed using Inveon Research Workplace (Siemens, Knoxville, TN) and the Matlab program. Partial volume correction was performed following an established method to eliminate the spillover of signals from tissue outside the brain50. Decay correlation was applied to compensate for the decay of 64Cu over time. The pixel intensity of the PET image was quantified as percent injected dose per gram tissue (%ID/g).

Analysis of the correlation between PCI and PET

Since the ultrasound imaging probe was co-axially aligned with the FUS transducer, the focal point of the FUS transducer was located in the ultrasound imaging plane. To investigate the correlation between PCI and PET, we first identified the PET voxel location with the highest radioactivity inside the brain, which was considered to be the location of the FUS focus. Then the PET images acquired in the coronal plane passing through this voxel was selected and considered to be the corresponding imaging plane for the B-mode/PCI. This selection of the corresponding PET imaging plane was verified in all the cases as the same anatomic features of the mouse skulls were observed in the B-mode images and corresponding CT images (Fig. 1). Then six controlling points were selected on the B-mode and corresponding CT images (Fig. 1). The CT images were translated and rotated to fit the same controlling points on B-mode images. Through this process, the transformation matrix was acquired and applied to PET images to register the PET images with PCI images.

For each mouse, the capability of SCD in predicting 64Cu-AuNC delivery location was characterized by quantifying the offsets between the pixel location of the SCD max and the location with the maximum 64Cu-AuNC concentration. The offsets of these two locations were decomposed to the lateral direction and axial direction. The capability of SCD in predicting 64Cu-AuNCs concentration was determined by evaluating the pixel-by-pixel correlation between the SCD map and the concentration map. The pixel size of the PET scanner was 0.8 mm × 0.8 mm, corresponding to the intrinsic sampling size of the scanner. The SCD maps had a pixel size of 0.2 mm × 0.2 mm. To perform a pixel by pixel comparison between these two maps, we first aligned the two images by aligning the pixel location of SCD max and the pixel with the highest concentration. Then we decreased the pixel size of the SCD map by downsampling to match that of the PET. The correlations between these two maps within the 2D region (Fig. 5A) were evaluated by the segmented linear regression using GraphPad Prism (Version 6.04, La Jolla, CA, USA), and the goodness of fit was assessed by the correlation coefficient, R2.

Ex vivo quantification

To validate the correlation found between PCI and in vivo PET imaging, nine additional mice were used to perform ex vivo quantification of 64Cu-AuNCs radioactivity using gamma counting and Au concentration by ICP-MS20. These mice were treated using the same FUS treatment protocol as described in 4.1. After FUS treatment, they were sacrificed by transcardial perfusion at 24 h after FUS treatment; their brains were collected and sliced coronally into 2-mm slices. Then the slices containing the brainstem was cut into two halves (left and right representing FUS-treated and non-treated samples, respectively) and the radioactivity was counted using a Beckman 8000 gamma counter (Beckman, Fullerton, CA). The count rate (counts per minute, CPM) for each tissue sample was corrected by automatic background subtraction. Decay correction was applied (compensated for the decay of 64Cu radioactivity over time). The corrected CPM from each tissue sample was normalized both to the mass of the tissue sample (in grams, g) and to the injected dose (ID). The concentration of 64Cu-AuNCs in each tissue sample was then calculated as %ID/g. The correlation between the radioactivity (%ID/g) of 64Cu-AuNCs in the FUS-treated and contralateral non-treated brainstem and the corresponding spatial-averaged SCD within the same regions was evaluated using the segmented linear regression.

After the gamma counting, these samples were then digested using a high-pressure microwave digestion system (Milestone Inc. Monroe, CT) and the gold concentrations in the digested brain tissue samples were determined using ICP-MS (Elan DRC-e, PerkinElmer, Germany). Au standard was used to generate the standard curve. The Au concentration was expressed as the percentage of the Au in the brainstem over the total injected Au normalized by the tissue weight (%ID/g). The Au concentration (%ID/g) in FUS-treated and non-treated brainstem was correlated with the corresponding spatial-averaged SCD within the same regions using the segmented linear regression.