High-throughput ultrasound neuromodulation in awake and freely behaving rats.

Transcranial ultrasound neuromodulation is a promising potential therapeutic tool for the noninvasive treatment of neuropsychiatric disorders. However, the expansive parameter space and difficulties in controlling for peripheral auditory effects make it challenging to identify ultrasound sequences and brain targets that may provide therapeutic efficacy. Careful preclinical investigations in clinically relevant behavioral models are critically needed to identify suitable brain targets and acoustic parameters. However, there is a lack of ultrasound devices allowing for multi-target experimental investigations in awake and unrestrained rodents. We developed a miniaturized 64-element ultrasound array that enables neurointerventional investigations with within-trial active control targets in freely behaving rats. We first characterized the acoustic field with measurements in free water and with transcranial propagation. We then confirmed in vivo that the array can target multiple brain regions via electronic steering, and verified that wearing the device does not cause significant impairments to animal motility. Finally, we demonstrated the performance of our system in a high-throughput neuromodulation experiment, where we found that ultrasound stimulation of the rat central medial thalamus, but not an active control target, promotes arousal and increases locomotor activity.

Dual-Mode 1-D Linear Ultrasound Array for Image-Guided Drug Delivery Enhancement Without Ultrasound Contrast Agents.

Pulsed high-intensity focused ultrasound (pHIFU) uses nonlinearly distorted millisecond-long ultrasound pulses of moderate intensity to induce inertial cavitation in tissue without administration of contrast agents. The resulting mechanical disruption permeabilizes the tissue and enhances the diffusion of systemically administered drugs. This is especially beneficial for tissues with poor perfusion such as pancreatic tumors. Here, we characterize the performance of a dual-mode ultrasound array designed for image-guided pHIFU therapies in producing inertial cavitation and ultrasound imaging. The 64-element linear array (1.071 MHz, an aperture of 14.8×51.2 mm, and a pitch of 0.8 mm) with an elevational focal length of 50 mm was driven by the Verasonics V-1 ultrasound system with extended burst option. The attainable focal pressures and electronic steering range in linear and nonlinear operating regimes (relevant to pHIFU treatments) were characterized through hydrophone measurements, acoustic holography, and numerical simulations. The steering range at ±10% from the nominal focal pressure was found to be ±6 mm axially and ±11 mm azimuthally. Focal waveforms with shock fronts of up to 45 MPa and peak negative pressures up to 9 MPa were achieved at focusing distances of 38-75 mm from the array. Cavitation behaviors induced by isolated 1-ms pHIFU pulses in optically transparent agarose gel phantoms were observed by high-speed photography across a range of excitation amplitudes and focal distances. For all focusing configurations, the appearance of sparse, stationary cavitation bubbles occurred at the same P- threshold of 2 MPa. As the output level increased, a qualitative change in cavitation behavior occurred, to pairs and sets of proliferating bubbles. The pressure P- at which this transition was observed corresponded to substantial nonlinear distortion and shock formation in the focal region and was thus dependent on the focal distance of the beam ranging within 3-4 MPa for azimuthal F -numbers of 0.74-1.5. The array was capable of B-mode imaging at 1.5 MHz of centimeter-sized targets in phantoms and in vivo pig tissues at depths of 3-7 cm, relevant to pHIFU applications in abdominal targets.

Dual-Use Transducer for Ultrasound Imaging and Pulsed Focused Ultrasound Therapy.

Pulsed focused ultrasound (pFUS) uses short acoustic pulses delivered at low duty cycle and moderate intensity to noninvasively apply mechanical stress or introduce disruption to tissue. Ultrasound-guided pFUS has primarily been used for inducing cavitation at the focus, with or without contrast agents, to promote drug delivery to tumors. When applied in tandem with contrast agents, pFUS is often administered using an ultrasound imaging probe, which has a small footprint and does not require a large acoustic window. The use of nonlinear pFUS without contrast agents was recently shown to be beneficial for localized tissue disruption, but required higher ultrasound pressure levels than a conventional ultrasound imaging probe could produce. In this work, we present the design of a compact dual-use 1-MHz transducer for ultrasound-guided pFUS without contrast agents. Nonlinear pressure fields that could be generated by the probe, under realistic power input, were simulated using the Westervelt equation. In water, fully developed shocks of 42-MPa amplitude and peak negative pressure of 8 MPa were predicted to form at the focus at 458-W acoustic power or 35% of the maximum reachable power of the transducer. In absorptive soft tissue, fully developed shocks formed at higher power (760 W or 58% of the maximum reachable power) with the shock amplitude of 33 MPa and peak negative pressure of 7.5 MPa. The electronic focus-steering capabilities of the array were evaluated and found to be sufficient to cover a target with dimensions of 19 mm in axial direction and 44 mm in transversal direction.

Transcutaneous Ultrasound-Mediated Nonviral Gene Delivery to the Liver in a Porcine Model.

Ultrasound (US)-mediated gene delivery (UMGD) of nonviral vectors was demonstrated in this study to be an effective method to transfer genes into the livers of large animals via a minimally invasive approach. We developed a transhepatic venous nonviral gene delivery protocol in combination with transcutaneous, therapeutic US (tUS) to facilitate significant gene transfer in pig livers. A balloon catheter was inserted into the pig hepatic veins of the target liver lobes via jugular vein access under fluoroscopic guidance. tUS exposure was continuously applied to the lobe with simultaneous infusion of pGL4 plasmid (encoding a luciferase reporter gene) and microbubbles. tUS was delivered via an unfocused, two-element disc transducer (H105) or a novel focused, single-element transducer (H114). We found applying transcutaneous US using H114 and H105 with longer pulses and reduced acoustic pressures resulted in an over 100-fold increase in luciferase activity relative to untreated lobes. We also showed effective UMGD by achieving focal regions of >105 relative light units (RLUs)/mg protein with minimal tissue damage, demonstrating the feasibility for clinical translation of this technique to treat patients with genetic diseases.

Ultrasound-Mediated Gene Therapy in Swine Livers Using Single-Element, Multi-lensed, High-Intensity Ultrasound Transducers.

We have achieved significant enhancement of gene delivery into livers of large animals using ultrasound (US)-targeted microbubble (MB) destruction methods. An infusion of pGL4 (encoding a luciferase reporter gene) plasmid DNA (pDNA) and MBs into a portal-vein segmental branch of a porcine liver was exposed to US for 4 min. Therapeutic US induced cavitation of MBs to temporarily permeabilize the vascular endothelium and cell membranes, allowing entry of pDNA. We obtained a 64-fold enhancement in luciferase expression in pig livers compared to control without US using an unfocused, dual-element transducer (H105, center frequency [fc] = 1.10 MHz) at 2.7 MPa peak negative pressure (PNP). However, input electrical energy was limited, and modified transducers were designed to have spherical (H185A, fc = 1.10 MHz) or cylindrical foci (H185B, fc = 1.10 MHz; H185D, fc = 1.05 MHz) to enhance PNP output. The revised transducers required less electrical input to achieve 2.7 MPa PNP compared to H105, thereby allowing PNP outputs of up to 6.2 MPa without surpassing the piezo-material limitations. Subsequently, luciferase expression significantly improved up to 9,000-fold compared to controls with minor liver damage. These advancements will allow us to modify our current protocols toward minimally invasive US gene therapy.

Ultrasound-targeted microbubble destruction-mediated gene delivery into canine livers.

Ultrasound (US) was applied to a targeted canine liver lobe simultaneously with injection of plasmid DNA (pDNA)/microbubble (MB) complexes into a portal vein (PV) segmental branch and occlusion of the inferior vena cava (IVC) to facilitate DNA uptake. By using a 1.1 MHz, 13 mm diameter transducer, a fivefold increase in luciferase activity was obtained at 3.3 MPa peak negative pressure (PNP) in the treated lobe. For more effective treatment of large tissue volumes in canines, a planar unfocused transducer with a large effective beam diameter (52 mm) was specifically constructed. Its apodized dual element configuration greatly reduced the near-field transaxial pressure variations, resulting in a remarkably uniform field of US exposure for the treated tissues. Together with a 15 kW capacity US amplifier, a 692-fold increase of gene expression was achieved at 2.7 MPa. Transaminase and histology analysis indicated minimal tissue damage. These experiments represent an important developmental step toward US-mediated gene delivery in large animals and clinics.