Alzheimer’s Disease (AD) is a progressive neurodegenerative disease that accounts for 60-70% of the 55 million worldwide dementia cases. The blood-brain barrier (BBB) acts as a mediator between the brain and cerebral vasculature, preventing the passage of deleterious substances, albeit significantly reducing drug delivery efficiency to the brain. The BBB is comprised of specialized cells that both maintain brain homeostasis and respond to pathological stress. Focused ultrasound-induced blood-brain barrier opening (FUS-BBBO) presents a noninvasive, transient, and targeted method to enhance drug delivery by locally increasing BBB permeability, in addition to modulating the neuroimmune landscape. This technique offers countless therapeutic opportunities for diseases of the brain, especially neurodegenerative disorders and AD. Previous studies have demonstrated effective pathological amelioration and cognitive improvement by applying FUS-BBBO in severely progressed murine models of AD. However, growing interest in clinical translation of FUS-BBBO and in alternative, early intervention therapeutic paradigms necessitates a more thorough characterization of the role of FUS-BBBO in AD therapeutics, particularly at early disease states. This thesis focuses on characterizing three key elements of FUS-BBBO treatment for applications to AD therapy. First, the physical effects of age and AD on the brain’s response to a single session of FUS-BBBO will be characterized. Next, the extent of cognitive and pathological improvement resulting from early intervention in male and female AD mice with repeated FUS-BBBO alone, then in combination with an amyloid-targeting therapeutic will be evaluated. Finally, the cell-specific response of astrocytes, oligodendrocytes, neurons and endothelial cells to FUS-BBBO will be characterized to elucidate the contribution of these cell types to previously observed cognitive and pathological improvements in male and female, young and aged, wild-type and AD mice. Broadly, the findings of the work described herein will elucidate the role of FUS-BBBO in AD therapeutics. By defining the most important considerations for applying FUS-BBBO in aged and AD populations, characterizing the expected cognitive and pathological outcomes from early FUS-BBBO intervention, and characterizing a time course of cell-specific responses to elucidate the mechanisms that underlie these observations, these aims collectively seek to improve our understanding and optimize our use of FUS-BBBO for AD therapeutics.

Establishment of animal model for studying treatment efficacy of low-intensity pulsed ultrasound stimulations for accelerating bone-tendon repair. Standard partial patellectomy was conducted in the 18-week old rabbits that were then divided into the LIPUS treatment and control groups. The animals were followed for 2, 4, 8, and 16 weeks for various tissue analyses. LIPUS was applied to the experimental animals from postoperative day 3 to 16 weeks. We demonstrated that the healing process of PPT junction was initiated through endochondral ossification. The results showed that the size and length of newly formed bone, and its bone mineral content (BMC), but not its bone mineral density (BMD) were correlated with the failure load, ultimate strength and energy at failure. Using radiographic, biomechanical, histomorphologic and biomechanical methods, it was found that LIPUS had significant accelerating effect on PPT junction repair. We validated our study hypothesis in that LIPUS enhances bone-tendon junction healing by stimulating angiogenesis, chondrogenesis and osteogenesis. / Establishment of in vitro model for mechanism study on effects of low-intensity pulsed ultrasound stimulations. An in vitro model of osteoblast-like cell line (SaOS-2 cells) was studied using cDNA microarray to explore the molecular mechanism mediated by LIPUS. This microarray analysis revealed a total of 165 genes that were regulated at 4 and 24 hours by LIPUS treatment in osteoblastic-like cells. These genes belonged to more than ten protein families based on their function and were involved in some signal transduction pathways. This study has validated the hypothesis that LIPUS can regulate a number of critical genes transient expressions in osteoblast cell line Saos-2. / Keywords. partial patellectomy model; bone-tendon junction repair; low intensity pulsed ultrasound stimulations (LIPUS); gene expression; complementary DNA microarray; rabbit. / This study explored the intact morphology, regular healing and the augmented healing under the effects of low intensity pulsed ultrasound stimulations (LIPUS) on the patella-patella tendon (PPT) junction in a rabbit partial patellectomy model. To probe its possible mechanism, the key genes involved in regulating osteogenesis mediated by LIPUS were identified using the state-of-the-art methods---complementary DNA microarray. / Lu Hongbin. / "June 2006." / Advisers: Ling Qin; Kwok Sui Leung. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1548. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 259-288). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Treatment of brain diseases remains extremely challenging partly due to the fact that critical drug delivery is hindered by the blood-brain barrier (BBB), a specialized and highly selective barrier lining the brain vasculature. Focused ultrasound (FUS), combined with systematically administered microbubbles (MBs), has been established as a technique to noninvasively, locally, and transiently open the BBB. The primary mechanism for temporarily opening the BBB using FUS is microbubble cavitation, a phenomenon that occurs when the circulating microbubbles interact with the FUS beam in the brain vasculature. Over the past two decades, many preclinical and clinical applications of FUS-induced BBB opening have been developed, but certain challenges, such as drug delivery route, cavitation control, inflammation onset, and overall accessibility of the technology, have affected its efficient translation to the clinic. This dissertation focuses on optimizing three aspects of FUS-induced BBB opening for therapeutic applications. The first specific aim investigated FUS-induced BBB opening for drug delivery through the intranasal route. Optimal sonication parameters were determined and applied to FUS-enhanced intranasal delivery of neurotrophic factors in a Parkinson’s Disease mouse model. In the second specific aim, cavitation levels affecting the inflammatory response due to BBB opening with FUS were optimized. The relationship between cavitation during FUS-induced BBB opening and the local inflammation was examined, and a cavitation-based controller system was developed to modulate the inflammatory response. In the third specific aim, the devices used for FUS-induced BBB opening were streamlined. A conventional system for FUS-induced BBB opening includes two transducers: one for therapy and another for cavitation monitoring (single element) or imaging (multi-element). In this aim, a single linear array transducer capable of synchronous BBB opening and cavitation imaging was developed, creating a cost-effective and highly accessible “theranostic ultrasound” device. The feasibility of theranostic ultrasound (TUS) was demonstrated in vivo in both mice and non-human primates. In summary, the findings and methodologies in this dissertation optimized FUS-enhanced intranasal delivery across the BBB, developed a cavitation-controlled system to modulate inflammation in the brain, which has been advantageous in reducing pathology and designed a new system for theranostic ultrasound for drug delivery to the brain. Taken altogether, this thesis contributes to the efficient advancement and optimization of FUS-induced BBB opening technology, thus enhancing its clinical adoption in the fight to treat many challenging brain diseases.

Chronic wounds pose a major threat to healthcare systems, and can be caused by a variety of factors, from diabetes to battlefield injuries. Traditional wound care does not account for a patient’s specific circumstances, and is only effective in up to 50% of cases. As such, there is a growing need for smarter wound healing technologies that can be used in a wide array of settings, from low resource hospitals to at home, and that provide customized treatments that can be administered without trained professionals. In this dissertation, we detail the development of technologies for customized treatments to accelerate wound healing. In Aim 1, we used a water-activated, electronics-free dressing to accelerate wound healing in a diabetic mouse model. Electrical stimulation has previously been used to improve wound healing; however, common dressings often require that the wearer be physically connected to large benchtop electronics, are expensive to produce, and/or contain toxic elements. We demonstrated that this device, developed by our collaborators, accelerated time to wound closure by approximately 30%, on par with other, more complex devices, and further improved angiogenesis, collagen intensity, and reduced inflammation when compared with the controls. Furthermore, we demonstrated that this device is biocompatible and does not affect mouse behavior; the device does not heat up when activated, and did not impact the distance the mice traveled during a ten-minute measurement window. We are exploring additional use cases for this technology to further accelerate wound healing, including through iontophoresis, the use of electric currents to transmit drugs through the skin. In Aim 2, we developed ultrasound-responsive, perfluorocarbon-based nanoparticles for spatiotemporal control of payload release. Focused ultrasound can be used to selectively and noninvasively trigger perfluorocarbon vaporization, releasing the payload from the triggered nanoparticles without disrupting nearby nanoparticles. Furthermore, these systems can be designed to remain stable when not in use, and to then release their payloads at safe acoustic pressures. We developed PLGA-coated, perfluorocarbon-based nanoparticles of various sizes, geometries, and with payloads. We used B-mode imaging and acoustic signal analysis to determine the acoustic thresholds of these nanoparticles, and then incorporated the nanoparticles into gels, from which we measured their payload release upon exposure to focused ultrasound. Initial in vivo testing of these nanoparticles showed that they remained stable until application of focused ultrasound. Such a technology has the potential to customize healing treatments, releasing specific payloads when and where they are most needed. In Aim 3, we integrated components of a closed-loop wound healing system in vitro and in vivo. This system comprises an ultrasound bandage to provide both sensing of the wound and treatment to the wound, sensors to analyze the wound state, drug delivery depots to selectively release payload into the wound, and a machine learning algorithm to guide treatment based on sensor values. We developed ultrasound-responsive microcapsules to selectively release drugs, and tested these in vitro and in a diabetic mouse wound model. We tested the ultrasound-responsiveness of alginate-acrylamide hydrogels in vitro and in vivo. We additionally tested versions of the ultrasound bandage and lactate sensors in vivo, and tested various combinations of these technologies. We tested the release of growth factor from the hydrogels using focused ultrasound while collecting ultrasound images for analysis, and demonstrated that a commercial-grade ultrasound probe can differentiate between wound healing states, which suggests that this technology will be translatable beyond the lab. Future work could demonstrate a truly closed-loop system, and could move beyond the diabetic mouse model, to one more similar to healing in humans. In this dissertation, we demonstrated technologies that can, individually or together, be used to improve wound healing in a variety of settings. Overall, this work advances the field of wound healing and demonstrates a suite of tools that can be used to provide customized treatments based on a patient’s needs, towards a vision for closed-loop wound healing systems.

Over 50 million people globally suffer from neurodegenerative disorders—a number that is steadily increasing as the general population ages. Yet, effective treatments for neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD) remain limited, primarily due to the presence of a natural protective biological barrier lining cerebral blood vessels called the blood-brain barrier (BBB). The blood- brain barrier prevents passage of nearly 98% of small molecules from blood vessels to brain tissue, while most therapies designed for neurodegenerative disorders, such as gene therapies, are considered large-molecule drugs, making development of efficacious pharmacological treatments extremely challenging. Present strategies to bypass the BBB for drug delivery broadly fall into two categories: non-invasive but non-targeted methods, or targeted but invasive surgical procedures such as direct intracranial injection. Currently, the only method poised clinically to provide both non-invasive and targeted drug delivery to the brain is focused ultrasound (FUS). When combined with intravenously administered ultrasound contrast agents called microbubbles which oscillate within blood vessels in response to FUS pressure waves, FUS can safely and reversibly open the blood-brain barrier (BBB) in a highly targeted manner. This enhances drug delivery to brain regions affected by neurodegenerative disorders through a physical mechanism known as acoustic cavitation. A majority of FUS research to date has centered around development and clinical translation of stereotactic FUS guided by magnetic resonance imaging (MRI) for treatment monitoring, commonly referred to as MRgFUS. However, MRgFUS exhibits cost, accessibility, and portability barriers to implementation in medical centers globally. Alternatively, our group has developed cost-effective and accessible ultrasound-guided FUS (USgFUS) configurations, which have the potential to enable BBB opening and drug delivery treatment outside of an MRI with treatment guidance facilitated by neuro-navigation technology and cavitation monitoring. While most USgFUS systems developed prior to this dissertation achieve therapeutic opening of the BBB and cavitation monitoring with separate ultrasound transducers, this thesis focuses primarily on development and optimization of a single-transducer technique for both therapy and monitoring called theranostic ultrasound (ThUS). In Aim 1, we show that a repurposed diagnostic ultrasound array reprogrammed with focused imaging pulses can produce therapeutically relevant ultrasound energy through primate skulls, and can induce multi-site modulatory drug and gene delivery depending on the ThUS parameters applied. In Aims 2 and 3, we apply ThUS-mediated drug and gene delivery for pre-clinical neuroscience and therapeutic applications in PD, respectively. In Aim 2, we demonstrated non-invasive delivery of specialized genes and nanoparticles which together enable remote stimulation and recording of neuronal activity, a synergistic process which could enable remote brain-to-brain communication. In Aim 3, we leveraged ThUS-mediated gene therapy to restore degenerated neurons in a PD mouse model, achieving nearly 85% restoration of diseased dopaminergic neurons non-invasively. Finally, in Aim 4, we translated ThUS-mediated BBB opening to non-human primates (NHP) to determine initial feasibility of targeted gene expression facilitated by a low frequency, custom ThUS array. We demonstrated that both conventional USgFUS and ThUS configurations can safely induce targeted gene expression in brain regions implicated in PD in rhesus macaques, motivating translation of USgFUS for gene therapy in the clinic. The aims in this dissertation collectively underscore the growing number of pre-clinical applications which could benefit from ThUS technology, while propelling USgFUS methodologies as a whole to the brink of clinical translation for unprecedented access to efficacious non-invasive gene therapy for neurodegenerative disorders in the future.

Elastography, which is based on applying pressure and estimating the resulting deformation, involves the forward problem to obtain the strain distributions and inverse problem to construct the elastic distributions consistent with the obtained strains on observation points. This thesis focuses on the former problem whose solution is used as an input to the latter problem. The aim is to provide the inverse problem community with accurate strain estimates of a coronary artery vessel wall. In doing so, a new ultrasonic image-based elastography approach is developed. Because the accuracy and quality of the estimated strain fields depend on the resolution level of the ultrasound image and to date best resolution levels obtained in the literature are not enough to clearly see all boundaries of the artery, one of the main goals is to acquire high-resolution coronary vessel wall ultrasound images at different pressures. For this purpose, first an experimental setup is designed to collect radio frequency (RF) signals, and then image formation algorithm is developed to obtain ultrasound images from the collected signals. To segment the noisy ultrasound images formed, a geodesic active contour-based segmentation algorithm with a novel stopping function that includes local phase of the image is developed. Then, region-based information is added to make the segmentation more robust to noise. Finally, elliptical deformable template is applied so that a priori information regarding the shape of the arteries could be taken into account, resulting in more stable and accurate results. The use of this template also implicitly provides boundary point correspondences from which high-resolution, size-independent, non-rigid and local strain fields of the coronary vessel wall are obtained.