Focused ultrasound in conjunction with the systemic administration of microbubbles has been shown to open the bloodbrain barrier (BBB) selectively, noninvasively and reversibly. In this study, we investigate the dependence of the BBB opening's reversibility on the peak-rarefactional pressure (0.30-0.60 MPa) as well as the microbubble size (diameters of 1-2, 4-5, or 6-8 mm) in mice using contrast-enhanced T 1 -weighted (CE-T 1 ) MR images (9.4 T). Volumetric measurements of the diffusion of Gd-DTPA-BMA into the brain parenchyma were used for the quantification of the BBB-opened region on the day of sonication and up to 5 days thereafter. The volume of opening was found to increase with both pressure and microbubble diameter. The duration required for closing was found to be proportional to the volume of opening on the day of opening, and ranged from 24 h, for the smaller microbubbles, to 5 days at high peak-rarefactional pressures. Overall, larger bubbles did not show significant differences. Also, the extent of BBB opening decreased radially towards the focal region until the BBB's integrity was restored. In the cases where histological damage was detected, it was found to be highly correlated with hyperintensity on the precontrast T 1 images. Magn Reson Med 67:769-777,

Ultrasound methods in conjunction with microbubbles have been used for brain drug delivery, treatment of stroke, and imaging of cerebral blood flow. Despite advances in these areas, questions remain regarding the range of ultrasound parameters that disrupt the blood-brain barrier (BBB). In this study, several conditions were investigated to either enhance or reduce the likelihood of BBB disruption. Pulsed focused ultrasound (frequency: 1.5 MHz, pressure: 0.46 MPa, pulse repetition frequency (PRF): 0.1 to 25 Hz, pulse length (PL): 0.03 to 30 milliseconds) was noninvasively and locally administered to a predetermined region in the left hemisphere in the presence of circulating preformed microbubbles (Definity, Lantheus Medical Imaging, N. Billerica, MA, USA; 0.01, 0.05, 0.25 μL/g). Trans-BBB delivery of 3-kDa dextran was observed at PRFs as low as 1 Hz, whereas consistent delivery was observed at 5 Hz and above. Delivery was demonstrated at a PL as low as 33 microseconds. Although the delivered dextran concentration increased with the PL, this also increased the heterogeneity of the resulting distribution. In conclusion, key parameters that disrupt the BBB were identified out of a wide range of conditions. Reducing the total number of emitted acoustic cycles by shortening the PL, or decreasing the PRF, was also found to facilitate a more spatially uniform distribution of delivered dextran.

Recombinant adeno-associated virus (rAAV) has shown great promise as a potential cure for neurodegenerative diseases. The existence of the blood-brain barrier (BBB), however, hinders efficient delivery of the viral vectors. Direct infusion through craniotomy is the most commonly used approach to achieve rAAV delivery, which carries increased risks of infection and other complications. Here we report a focused ultrasound (FUS) facilitated, non-invasive rAAV delivery paradigm that is capable of producing targeted and neuron-specific transductions. Oscillating ultrasound contrast agents (i.e. microbubbles), driven by focused ultrasound waves, temporarily “unlocking” the BBB, allowing the systemically administrated rAAVs to enter the brain parenchyma, while maintaining their bioactivity and selectivity. Taking the advantage of the neuron-specific promoter-synapsin, rAAV gene expression was triggered almost exclusively (95%) in neurons of the targeted (i.e. caudate-putamen) region. Both behavioral assessment and histological examination revealed no significant long term adverse effects (in the brain and several other critical organs) for this combined treatment paradigm. Results from this study demonstrated the feasibility and safety for the non-invasive, targeted rAAV delivery technique, which might have provided a new arena for gene therapy in both pre-clinical and clinical settings.

The most challenging aspect of intravenously-administered drugs currently developed to treat central nervous system (CNS) diseases is their impermeability through the blood–brain barrier (BBB), a specialized vasculature system protecting the brain microenvironment. Focused ultrasound (FUS) in conjunction with systemically administered microbubbles has been shown to open the BBB locally, noninvasively, and reversibly. The objective of this study was to investigate the effect of FUS (center frequency: 1.5 MHz) pulse length (PL), ranging here from 67 µs to 6.7 ms, on the physiology of the FUS-induced BBB opening. Dynamic contrast-enhanced (DCE) and T1-weighted magnetic resonance imaging (MRI) were used to quantify the permeability changes using transfer rate (Ktrans) mapping, the volume of BBB opening (VBBB) and the reversibility timeline of the FUS-induced BBB opening, with the systemic administration of microbubbles at different acoustic pressures, ranging from 0.30 to 0.60 MPa. Permeability and volume of opening were both found to increase with the acoustic pressure and pulse length. At 67-µs PL, the opening pressure threshold was 0.45 MPa, with BBB opening characteristics similar to those induced with 0.60 MPa at the same PL, as well as with 0.67-ms PL/0.30 MPa. On average, these cases had Ktrans = 0.0049 ± 0.0014 min−1 and VBBB = 3.7 ± 4.3 mm3, and closing occurred within 8 h. The 6.7-ms PL/0.30 MPa induced similar opening with 0.67-ms PL/0.45 MPa, and a closing timeline of 24 to 48 h. On average, Ktrans was 0.0091 ± 0.0029 min−1 and VBBB was 14.13 ± 7.7 mm3 in these cases. Also, there were no significant differences between the 6.7-ms PL/0.45 MPa, 0.67-ms PL/0.60 MPa and 6.7-ms PL/0.60 MPa cases, yielding on average a Ktrans of 0.0100 ± 0.0023 min−1 and VBBB equal to 20.1 ± 5.7 mm3. Closing occurred within 48 to 72 h in these cases. Stacked histograms of the Ktrans provided further insight to the non-uniform spatial distribution of permeability changes and revealed a correlation with the closing timeline. These results also suggest a beneficial complementary relationship between the elongation of the PL and the decrease of the peak negative acoustic pressures, and vice versa. Linear regression between Ktrans and VBBB showed a good correlation fit. Also, the time required for closing linearly increased with VBBB. The volume rate of decrease was measured to be 11.4 ± 4.0 mm3 per day, suggesting that the closing timeline could be predicted from the initial volume of opening. Finally, no histological damage was detected in any of the cases 7 d post-FUS, indicating the safety of the methodology and parameters used.

The brain-derived neurotrophic factor (BDNF) has been shown to have broad neuroprotective effects in addition to its therapeutic role in neurodegenerative disease. In this study, the efficacy of delivering exogenous BDNF to the left hippocampus is demonstrated in wild-type mice (n=7) through the noninvasively disrupted blood-brain barrier (BBB) using focused ultrasound. The BDNF bioactivity was found to be preserved following delivery as assessed quantitatively by immunohistochemical detection of the pTrkB receptor and activated pAkt, pMAPK, and pCREB in the hippocampal neurons. It was therefore shown for the first time that systemically administered neurotrophic factors can cross the noninvasively disrupted BBB and trigger neuronal downstream signaling effects in a highly localized region in the brain. This is the first time that the administered molecule is tracked through the blood-brain barrier (BBB) and localized in the neuron triggering molecular effects. Additional preliminary findings are shown in wild-type mice with two additional neurotrophic factors such as the glia-derived neurotrophic factor (GDNF) (n=12) and neurturin (NTN) (n=2). This further demonstrates the impact of FUS for the early treatment of CNS diseases at the cellular and molecular level and strengthens its premise for FUS-assisted drug delivery and efficacy.