Transcranial Therapy for Intracerebral Hemorrhage and Other Brain Pathologies using Histotripsy

Brain pathologies including stroke and cancer are a major cause of death and disability. Intracerebral hemorrhage (ICH) accounts for roughly 12% of all strokes in the US. ICH is characterized by the rupture of vessels resulting in bleeding and clotting inside the brain. The presence of the clot causes immediate damage to surrounding brain tissue via mass effect with delayed toxic effects developing in the following days. This leads to 30-day mortality rate of 40% and motivates the need to quickly evacuate the clot. Craniotomy surgery and minimally invasive methods using thrombolytics are common procedures but are limited by morbidity and susceptibility to rebleeding, leading to poor outcomes. Histotripsy is a non-thermal ultrasound ablation technique that uses short duration, high amplitude rarefactional pulses (>26 MPa) delivered via an extracorporeal transducer to generate targeted cavitation using the intrinsic gas nuclei in the target tissue. The rapid and energetic bubble expansion and collapse of cavitation create high stress and strain in tissue at the focus that fractionate it into an acellular homogenate. This dissertation presents the role of histotripsy as a novel ultrasound technology with potential to address the need for an effective transcranial therapy for ICH and other brain pathologies. The first part of this work investigates the effects of ultrasound frequency and focal spacing on transcranial clot liquefaction using histotripsy. Histotripsy pulses were delivered using two 256-element hemispherical transducers of different frequency with 30-cm aperture diameters. Treatment durations ranged from 0.9-42.4 min. Liquefied clot volumes ranging from 6-59 mL were drained via catheter and syringe. The second part addresses safety concerns for histotripsy ICH treatment through investigation in a porcine ICH model. 1.75-mL clots were formed in the porcine brain. The cores of the clots were liquefied with histotripsy 48-h after formation, and the liquefied contents were either evacuated or left within the brain. A control group was left untreated. The cores of clots were liquefied without damage to the perihematomal tissue. An average volume of 0.9±0.5 mL was drained after histotripsy treatment. The third part presents the development of a catheter hydrophone method for transcranial phase aberration correction and drainage of the clot liquefied with histotripsy. A prototype hydrophone was fabricated to fit within a catheter. Corrections with the catheter hydrophone resulted in improvements in focal pressure of up to 60% at the geometric focus and 27%-62% across a range of electronic steering locations. The cores of clots liquefied with histotripsy were readily drained via the catheter. The fourth part focuses on the development of a preclinical system for translation to human cadaver ICH models. A 360-element, 700 kHz hemispherical array with a 30-cm aperture was designed and integrated with a surgical navigation system. Calibrated simulations of the transducer suggest an effective therapeutic volume between 48-105 mL through the human skull. The navigation system allows real-time targeting and placement of the catheter hydrophone via a pre-operative CT or MRI. The fifth part of this work extends transcranial histotripsy therapy beyond ICH to the treatment of glioblastoma. This section presents results from an investigation into cancer immunomodulation using histotripsy in a mouse glioblastoma model. The results suggest histotripsy has some immunomodulatory capacity as evidenced by a 2-fold reduction in myeloid derived suppressor cells and large increases in interferon-γ concentrations (3500 pg/mL) within the brain tumors of mice treated with histotripsy.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/155219/1/tgerhard_1.pd

Design and development of magnetic resonance imaging (MRI) compatible tissue mimicking phantoms for evaluating focused ultrasound thermal protocols

Animal models are often used to test the efficacy and safety of clinical applications employing focused ultrasound that range in various stages of research, development and commercialization. The animals are usually subjected to conditions that cause pain, distress and euthanasia. Access to cadaveric models is not easy and affordable for all research institutions, whereas conservation and changes of their physical properties over time can be a delimiting factor for translational research. The above set the motivation for this project, which its primary objective is to design and develop appropriate tissue mimicking phantoms using a simplistic and cost effective methodology. These phantoms are expected to contribute in reducing the need for animal testing and allow researchers to get hands experience with tools that will promote and accelerate testing in focused ultrasound thermal protocols. The main requirements for these phantoms are to be geometrically accurate, compatible with magnetic resonance imaging (MRI) and to be composed of materials that approximate the acoustic and thermal properties of the replicated tissues. Throughout the duration of the project three ultrasonic composite phantoms (head, femur bone-muscle and breast-rib) were developed. The acoustic properties of candidate materials were assessed using pulse-echo immersion and through transmission techniques. The thermal properties were estimated by observing the rate of heat diffusion following a sonication in the soft tissue parts with MR thermometry. Acrylonitrile butadiene styrene (ABS) was used to replicate bone tissue, where its acoustic attenuation coefficient was found to be 16.01 ± 6.18 dB/cm at 1 MHz and the speed of sound at 2048 ± 79 m/s. Soft tissue parts consisted out of agar-based gels doped with varying concentrations of additives that controlled the relative contribution of acoustic absorption (evaporated milk) and scatter (silica dioxide) to total attenuation independently. Brain tissue phantom (2 % w/v agar - 1.2 % w/v SiO2 - 25 % v/v evaporated milk) matched an attenuation coefficient of 0.59 ± 0.05 dB/cm-MHz whereas muscle and breast mimicking phantom (2 % w/v agar - 2 % w/v SiO2 - 40 % v/v evaporated milk) were estimated of inducing an attenuation coefficient of the order of 0.99 ±0.08 dB/cm-MHz. The speed of sound for the brain and muscle/breast recipe were estimated at 1485 ± 12 m/s and 1529 ± 13 m/s respectively. The thermal conductivity of the brain phantom was estimated to be 0.52 ± 0.06 W/mº-C and 0.57 ± 0.10 W/mº-C for the muscle/breast phantom. The acoustic and thermal properties of candidate materials were within range of the replicated tissues extracted from literature, except the speed of sound in ABS compared which was lower compared to bone (~3000 m/s). Three dimensional models of bone parts (skull, femur, rib) were reconstructed in Standard Tessellation Language (STL) format by segmenting bony tissue of interest from adult human computed tomography (CT) images. The STL bone models were 3D printed in ABS using a fused deposition modelling (FDM) machine. The final composite phantoms were fabricated by molding the agar based soft tissue phantoms inside/around the ABS bone phantoms. The functionality of all three composite phantoms was assessed with focused ultrasound sonications applied by a 1 MHz single element transducer while temperature was monitored with 1.5 Tesla MRI scanner. A spoiled gradient recalled (SPGR) pulse sequence was used to produce phase images that were analyzed using a custom coded software developed in Matlab that employed proton-resonance frequency shift (PRFS) thermometry

In vitro and in vivo investigation of enzymatic clot lysis through non-thermal mechanisms using focused ultrasound waves as an adjunct to thrombolytic drug tenecteplase and in synergy with microbubbles

In this thesis, the beneficial effect of ultrasound (US) energy is utilized for accelerating thrombolysis efficacy through non-thermal mechanisms. The outcome of the research, can be applied in the future for clinical use, providing alternative techniques (either noninvasive or invasive), for treating ischaemic stroke under MR guidance. To study the effect of US on thrombolysis efficacy, three different in vitro circulating flow clot models were developed. Fully retracted porcine blood clots were treated with US waves as an adjunct to thrombolytic drug tenecteplase (TNK-tPA), in the presence or absence of microbubbles (MBs). Each one of the flow clot models used, was designed to reproduce a different physiologic situation of middle cerebral artery (MCA) occlusion, since it is the most common cause of stroke. In all the proposed treatment protocols, temperature elevation at beam focus never exceeded 1 0C, providing that the contribution of thermal mechanisms to clot lysis was negligible. The first model, was reproducing a deep-seated MCA occlusion in a brain tissue. To provide a more realistic clinical environment, the study was conducted into a brain tissue mimicking phantom. Flow rate was set to 20 % of the maximum value occur in an open MCA and TNK-tPA dose was not exceeding the 30 % of the average maximum concentration in blood. Using 1.18 MHz focused US (FUS) waves, various experimental parameters that influence thrombolysis efficacy, were optimized. Maximum thrombolysis efficacy was observed when FUS pulses were used as an adjunct to thrombolytic drug in the presence of MBs. With this technique, 370 mg of clot mass was removed in 30 min, which may not be enough to achieve significant clinical benefits. However, further improvement was done (increase of flow rate and TNK-tPA concentration), to enhance thrombolysis efficacy. The second model, was reproducing a MCA occlusion occurred superficially. Flow rate was increased four times and TNK-tPA concentration was doubled. The optimum operating mode parameters obtained before, were employed in this study in order to investigate the impact of frequency and acoustic intensity on thrombolysis efficacy. Study findings established that higher FUS frequencies (1.18 MHz), are associated with enhanced thrombolysis compared to lower frequencies (0.6 MHz). Also, a linear increasing dependence between acoustic intensity and thrombolysis efficacy was observed. After 30 min of treatment with 1.18 MHz FUS exposures in synergy with TNKtPA and MBs, 1050 mg of clot mass was removed, which should be sufficient for timely recanalization of an occluded cerebral artery. The third model, was reproducing a MCA occlusion, treated invasively with a catheter-directed US device. The efficacy of two small planar ultrasonic transducers (operating at frequencies 3.7 and 5.2 MHz respectively), on clot lysis was evaluated. The values of flow rate and TNK-tPA concentration were kept the same with those used in the previous study. Using 3.7 MHz US waves in association with TNK-tPA and MBs for 30 min, 700 mg of clot mass was removed, showing that potentially, intravascular sonothrombolysis with such a transducer can be an effective method for treating stroke. Finally, the knowledge gained in vitro for enhancing TNK-tPA induced thrombolysis, was translated in vivo using an animal model. A blood clot was artificially formed into the right carotid artery of a rabbit, mimicking a MCA occlusion in humans. The complete flow blockage as well as the recanalization procedure were monitored using a 3D time of flight MR angiography. This novel technique, clearly demonstrated that the combination of 1.18 MHz FUS pulses with MBs, strongly accelerated the action of TNKtPA, leading through non-thermal mechanisms to full artery recanalization within 19 min

Evidence for the use of ultrasound therapy for the management of mandibular osteoradionecrosis

Introduction: The treatment of mandibular osteoradionecrosis includes antibiotics and curettage, hyperbaric oxygen, surgery, and more recently, therapeutic ultrasound. The aim of this thesis was to establish the possible mechanisms of action of therapeutic ultrasound, that could explain its excellent clinical results. Material and Methods: Two ultrasound machines were evaluated, a 'traditional' (1 MHz and 3 MHz) and a 'long wave' machine (45 kHz). Ultrasound was applied to human mandibular osteoblasts, gingival fibroblasts, peripheral blood monocytes (PBMc) and mice calvaria. The following in vitro assays were performed: cell proliferation, collagen and non-collagenous protein (NCP) synthesis, bone resorption, cytokines and angiogenesis factors production using ELISA and RT-PCR techniques, and nitric oxide production. To evaluate the effects of ultrasound on angiogenesis in vivo, the chick chorioallanlbic membrane assay (CAM) was used. The use of near infrared spectroscopy (NIRS) for the measurement of radiotherapy effects in the mandible (deoxyhaemoglobin concentrations) was also evaluated. Results: Ultrasound stimulated bone formation in the mice calvaria. Cell proliferation assays showed an increase of DNA synthesis in fibroblasts and osteoblasts, up to 52%. Collagen/NCR synthesis was also enhanced, in fibroblasts up to 48%, and in osteoblasts up to 112%. Bone resorption, part of the bone turnover process, was promoted, and there is suggestion that the cyclo-oxygenase pathway is involved. In relation to cytokine production, a slight stimulation of IL-1beta was noted in all cell types. There was no difference in IL-6 and TNFalpha levels. The angiogenesis factors, IL-8 and bFGF, were significantly stimulated in osteoblasts, and VEGF was significantly stimulated in fibroblasts, osteoblasts and PBMc. RT-PCR showed that ultrasound induces mRNA transcription for several cytokines and bone related proteins, with the most evident effect being the induction of VEGF transcription in osteoblasts. The CAM assay showed that direct ultrasound application and insonated medium from fibroblasts induced angiogenesis in vivo. The best overall stimulatory intensities were 15 and 30 mW/cm2(SA) with 45 kHz ultrasound, and 0.1 and 0.4 W/cm2(SAPA) with 1 MHz ultrasound. The NIRS evaluation showed that it is very sensitive to measure deoxyhaemoglobin concentrations, however these measurements are not reproducible. No age correlations could be performed, and the differences between normal and radiotherapy mandibles was not significant because of the great variability in the measurements. Conclusions: These results show that ultrasound can correct hypocellularity, hypoxia and hypovascularity observed in osteoradionecrosis. It stimulates cell proliferation, bone formation, healing, and angiogenesis. Further in vivo experiments are recommended as well as prospective clinical trials using therapeutic ultrasound for the treatment and prevention of osteoradionecrosis, but NIRS cannot be used to measure the outcome of treatment. Therapeutic ultrasound is a viable option for the management of mandibular osteoradionecrosis, since it is effective, inexpensive and readily available

Applications of Ultrasound-targeted Microbubble Cavitation with Sodium Nitrite and Nitro-alkenes

Microvascular obstruction is a common repercussion of percutaneous coronary intervention in treating acute myocardial infarction, and results from a combination of downstream microembolization, ischemia-reperfusion injury, and inflammation. Ultrasound-targeted microbubble cavitation (UTMC) uses external therapeutic ultrasound pulsation to target intravascularly infused microbubble contrast agents to produce shear stresses which mechanically disrupt obstructing microemboli. This work aims to enhance the therapeutic effects of UTMC through synergistic co-administration of nitrite for enhancement of perfusion and nitric oxide bioavailability (Aim 1) and development of a novel microbubble agent using nitro-alkenes for therapeutic reduction of inflammation after ischemia-reperfusion injury (Aim 2). For characterization and optimization of nitrite co-therapy with UTMC, a rat gastrocnemius model was used with contrast-enhanced ultrasound imaging. A nitric oxide porphyrinic membrane catheter probe was inserted into the treatment site for real-time measurement of nitric oxide concentration changes. In addition to nitrite co-therapy, effects of administering an endothelial nitric oxide synthase inhibitor as well as varying microbubble concentration and therapeutic ultrasound pressure were studied. Results showed that UTMC and nitrite demonstrated positive synergy for enhancing nitric oxide concentration and perfusion which depended on functional endothelial nitric oxide synthase. After a novel nitro-alkene microbubble agent was synthesized and characterized, it was applied in both healthy and ischemia-reperfusion injury rat gastrocnemius models. Tissue samples were collected after treatment for quantification of nitro-alkene delivery, changes in inflammatory gene expression, and contrast-enhanced ultrasound imaging was used to quantify changes in hindlimb perfusion after treatment. Results showed that incorporation of the nitro-alkene into a microbubble formulation with UTMC greatly enhanced targeted tissue delivery of the nitro-alkene compared to co-infusion with standard microbubbles and systemic infusion alone. In addition, nitro-alkene microbubble UTMC resulted in greatly enhanced perfusion as well as decreases in inflammatory gene expression. In all, both of these applications of enhancing UTMC therapy demonstrate significant mechanistic interactions with endogenous nitric oxide metabolism and may serve to enhance nitric oxide bioavailability through different pathways. They may also improve the relevance of UTMC in treating the biological sequelae of microvascular obstruction without compromising its mechanical function in disrupting microemboli