The impact of pulse repetition frequency on microbubble activity and drug delivery during focused ultrasound-mediated blood-brain barrier opening.

OBJECTIVE: Pulsed focused ultrasound (FUS) can deliver therapeutics to the brain by using intravenous microbubbles (MBs) to open the blood-brain barrier (BBB). MB emissions indicate treatment outcomes, like BBB opening (harmonics) and damage (broadband). Typically, a pulse repetition frequency (PRF) of 1 Hz is used, but the effect of PRF on MBs is not fully understood. We investigated the effect of PRF on MB activity and tracer delivery. Approach: The effect of PRF (0.125, 0.25, 0.5, 1, and 2 Hz) on MB activity was monitored through harmonic and wideband emissions during FUS sonications of the rat brain at 274.3 kHz. BBB opening was quantified through fluorescence imaging to estimate the concentration of Trypan Blue (TB) dye following a 75-pulse FUS exposure for PRFs of 1 and 0.25 Hz. Results: At a fixed acoustic pressure, the percentage change in maximum harmonic amplitude compared to the control (PRF = 1 Hz) decreased with increasing PRF, with a median change of 73.8% at 0.125 Hz and -38.3% at 2 Hz. There was no difference in the pressure threshold for broadband emissions between PRFs of 0.25 and 1 Hz. PRF = 0.25 Hz, led to a 68.2% increase in the mean concentration of TB measured after FUS, with a 53.9% increase in the mean harmonic sum, compared with PRF = 1 Hz. Harmonic emissions-based control at PRF = 0.25 Hz yielded similar TB delivery, with less damage at histology, compared with 1 Hz. Significance: For a fixed number of FUS pulses, reducing the PRF was shown to increase the magnitude of harmonic emissions and TB delivery, but not the threshold for broadband emissions. While further research is necessary to understand the mechanisms involved, these results may be useful to improve clinical safety margins and sensitivity to detecting small harmonic signals from cavitating MBs. .

A study combining microbubble-mediated focused ultrasound and radiation therapy in the healthy rat brain and a F98 glioma model.

Focused Ultrasound (FUS) has been shown to sensitize tumors outside the brain to Radiotherapy (RT) through increased ceramide-mediated apoptosis. This study investigated the effects of FUS + RT in healthy rodent brains and F98 gliomas. Tumors, or striata in healthy rats, were targeted with microbubble-mediated, pulsed FUS (220 kHz, 102-444 kPa), followed by RT (4, 8, 15 Gy). FUS + RT (8, 15 Gy) resulted in ablative lesions, not observed with FUS or RT only, in healthy tissue. Lesions were visible using Magnetic Resonance Imaging (MRI) within 72 h and persisted until 21 days post-treatment, indicating potential applications in ablative neurosurgery. In F98 tumors, at 8 and 15 Gy, where RT only had significant effects, FUS + RT offered limited improvements. At 4 Gy, where RT had limited effects compared with untreated controls, FUS + RT reduced tumor volumes observed on MRI by 45-57%. However, survival benefits were minimal (controls: 27 days, RT: 27 days, FUS + RT: 28 days). Histological analyses of tumors 72 h after FUS + RT (4 Gy) showed 93% and 396% increases in apoptosis, and 320% and 336% increases in vessel-associated ceramide, compared to FUS and RT only. Preliminary evidence shows that FUS + RT may improve treatment of glioma, but additional studies are required to optimize effect size.

Focused Ultrasound-Induced Blood-Spinal Cord Barrier Opening Using Short-Burst Phase-Keying Exposures in Rats: A Parameter Study.

Transient opening of the blood-spinal cord barrier has the potential to improve drug delivery options to the spinal cord. We previously developed short-burst phase-keying exposures to reduce focal depth of field and mitigate standing waves in the spinal canal. However, optimal short-burst phase-keying parameters for drug delivery have not been identified. Here, the effects of pressure, treatment duration, pulse length, burst repetition frequency and burst length on resulting tissue effects were investigated. Increased in situ pressures (0.23-0.33 MPa) led to increased post-treatment T1-weighted contrast enhancement in magnetic resonance imaging (p = 0.015). Increased treatment duration (120 vs. 300 s) led to increased enhancement, but without statistical significance (p = 0.056). Increased burst repetition frequency (20 vs. 40 kHz) yielded a non-significant increase in enhancement (p = 0.064) but corresponded with increased damage observed on histology. No difference was observed in enhancement between pulse lengths of 2 and 10 ms (p = 0.912), corresponding with a sharp drop in the recorded second harmonic signal during the first 2 ms of the pulse. Increasing the burst length from two to five cycles (514 kHz) led to increased enhancement (p = 0.014). Results indicate that increasing the burst length may be the most effective method to enhance drug delivery. Additionally, shorter pulse lengths may allow more interleaved targets, and therefore a larger treatment volume, within one sonication.

A Porcine Model of Transvertebral Ultrasound and Microbubble-Mediated Blood-Spinal Cord Barrier Opening.

Blood-spinal cord barrier opening, using focused ultrasound and microbubbles, has the potential to improve drug delivery for the treatment of spinal cord pathologies. Delivering and detecting ultrasound through the spine is a challenge for clinical translation. We have previously developed short burst, phase keying exposures, which can be used in a dual-aperture configuration to address clinical scale targeting challenges. Here we demonstrate the use of these pulses for blood-spinal cord barrier opening, in vivo in pigs. Methods: The spinal cords of Yorkshire pigs (n=8) were targeted through the vertebral laminae, in the lower thoracic to upper lumbar region using focused ultrasound (486 kHz) and microbubbles. Four animals were treated with a combination of pulsed sinusoidal exposures (1.0-4.0 MPa, non-derated) and pulsed short burst, phase keying exposures (1.0-2.0 MPa, non-derated). Four animals were treated using ramped short burst, phase keying exposures (1.8-2.1 MPa, non-derated). A 250 kHz narrowband receiver was used to detect acoustic emissions from microbubbles. Blood-spinal cord barrier opening was assessed by the extravasation of Evans blue dye. Histological analysis of the spinal cords was used to assess tissue damage and excised vertebral samples were used in benchtop experiments. Results: Ramped short burst, phase keying exposures successfully modified the blood-spinal cord barrier at 16/24 targeted locations, as assessed by the extravasation of Evans blue dye. At 4 of these locations, opening was confirmed with minimal adverse effects observed through histology. Transmission measurements through excised vertebrae indicated a mean transmission of (47.0 ± 7.0 %) to the target. Conclusions: This study presents the first evidence of focused ultrasound-induced blood-spinal cord barrier opening in a large animal model, through the intact spine. This represents an important step towards clinical translation.

Enhanced Detection of Bubble Emissions Through the Intact Spine for Monitoring Ultrasound-Mediated Blood-Spinal Cord Barrier Opening.

OBJECTIVE: We previously developed short burst, phase keying (SBPK) focused ultrasound (FUS) to mitigate standing waves in the human vertebral canal. Here, we show microbubble emissions from these pulses can be detected through the human vertebral arch and that these pulses are effective for blood-spinal cord barrier (BSCB) opening. METHODS: At f0 = 514 kHz, circulating microbubbles were sonicated through ex vivo human vertebrae (60 kPa-1 MPa) using a dual-aperture approach and SBPK exposures engineered to incorporate pulse inversion (PI). Signals from a 250 kHz receiver were analyzed using PI, short-time Fourier analysis and the maximum projection over the pulse train. In rats (n = 14), SBPK FUS+microbubbles was applied to 3 locations/spinal cord at fixed pressures (∼0.20-0.47 MPa). MRI and histology were used to assess opening and tissue damage. RESULTS: In human vertebrae between 0.2-0.4 MPa, PI amplified the microbubble/baseline ratio at f0/2 and 2f0 by 202 ± 40% (132-291%). This was maximal at 0.4 MPa, coinciding with the onset of broadband emissions. In vivo, opening was achieved at 40/42 locations, with mean MRI enhancement of 46 ± 32%(16%-178%). Using PI, f0/2 was detected at 14/40 opening locations. At the highest pressures (f0/2 present) histology showed widespread bleeding throughout the focal region. At the lowest pressures, opening was achieved without bleeding. CONCLUSION: This study confirmed that PI can increase sensitivity to transvertebral detection of microbubble signals. Preliminary in vivo investigations show that SBPK FUS can increase BSCB permeability without tissue damage. SIGNIFICANCE: SBPK is a clinically relevant pulse scheme and, in combination with PI, provides a means of mediating and monitoring BSCB opening noninvasively.

Analysis of Multifrequency and Phase Keying Strategies for Focusing Ultrasound to the Human Vertebral Canal.

Focused ultrasound has been shown to increase the permeability of the blood-brain barrier and its feasibility for opening the blood-spinal cord barrier has also been demonstrated in small animal models, with great potential to impact the treatment of spinal cord (SC) disorders. For clinical translation, challenges to transvertebral focusing of ultrasound energy on the human spinal canal, such as a focal depth of field and standing-wave formation, must be addressed. A dual-aperture approach using multifrequency and phase-shift keying (PSK) strategies for achieving a controlled focus in human thoracic vertebrae was investigated through numerical simulations and benchtop experiments in ex vivo human vertebrae. An ~85% reduction in the focal depth of field was achieved compared to a single-aperture approach at 564 kHz. Short-burst (two-cycle) excitations in combination with PSK were found to suppress the formation of standing waves in ex vivo human thoracic vertebrae when focusing through the vertebral laminae. The results make an important contribution toward the development of a clinical-scale approach for targeting ultrasound therapy to the SC.