Multisizer 3

FUS BBBO was performed with the RK-300 small bore FUS device (FUS Instruments, Toronto, CA). Heads of mice were shaved and depilated prior to supine placement and coupling to the transducer with degassed ultrasound gel. BBBO was performed with a 1.13 MHz single-element transducer using a 10 ms burst length over a 2000 ms period for 60 total sonications during a 2-min sonication duration. Fixed PNP application was performed using the “Burst” mode on the FUS Instruments software. PCD-modulated PNP was performed using the “Blood-brain Barrier” mode of the FUS Instruments software. Parameters used for this feedback control system included a starting pressure of 0.2 MPa, pressure increment of 0.05 MPa, maximum pressure of 0.4 MPa, 20 sonication baselines without microbubbles, AUC bandwidth of 500 Hz, AUC threshold of 10 standard deviations, pressure drop of 0.95, and frequency selection of the subharmonic, first ultraharmonic, and second ultraharmonic. Gadolinium contrast agent (Multihance) was injected as a bolus intravenously with a dose of 0.01 mmol diluted in saline at a molarity of 0.2 mmol/mL prior to T1-RARE image acquisition. Albumin-shelled microbubbles were made in-house as previously described^{59 (link)} and intravenously injected as a bolus dose of 10⁵ microbubbles per gram body weight. Distribution of microbubble diameter and concentration was acquired with a Coulter counter (Multisizer 3; Beckman Coulter, Fullerton, California) prior to sonication. High resolution T2-weighted images and T1-RARE images were used to guide FUS targeting to the pre-selected CCM. A single sonication target was used in all experiments, except in the case of PCD-modulated PNPs, in which two sonication targets were used. Mice receiving the repeat FUS BBBO regimens had all sonications staged 3 days apart with the same anatomical location targeted each time.

Samples (2 g) of subcutaneous adipose tissue were obtained inferior to the umbilicus after administration of 0.25 lidocaine with adrenaline (epinephrine) for local anesthesia, from which two 20–30-mg samples were used immediately for adipose cell-size distribution analysis by the osmium fixation, Beckman Coulter (Miami, FL) Multisizer III, curve-fitting analysis technique previously described (6 (link)). In addition to determining the fraction of large adipose cells (fraclarge) and the “peak diameter” of the large adipose cells as described, the “% of adipose cells above” (% large cells) and “% below” (% small cells) the nadir were calculated.
A secondary end point, the number of subcutaneous adipose cells, was estimated by the following formula: cell number = volume of subcutaneous abdominal adipose tissue/weighted volume per cell. Volume of adipose tissue was obtained from MRI scans, and average volume per cell was calculated as the weighted volume based on the relative number of cells per volume bin in the cell-volume histogram generated by the Multisizer software. We used the following formula: average volume per cell = Σ ⁴/₃π(^d_i/2)³p_i (that is, the sum of the volumes corresponding to each bin times the relative frequency (p) of that bin (i) (16 (link)). The number of large cells was then calculated by applying the percentage of large cells to the total number of cells.