The blood-brain barrier (BBB) is physiologically essential for brain homeostasis. While it protects the brain from noxious agents, it prevents almost all currently available drugs from crossing to the parenchyma. This greatly hinders drug delivery for the treatment of neurological diseases and disorders such as Parkinson’s, Alzheimer’s and Huntington’s, as well as the development of drugs for the treatment of such diseases. Current drug delivery techniques to the brain are either invasive and target specific, or non-invasive with low special specificity. Neither group of techniques are optimal for long term treatment of patients with neurological diseases or disorders. Focused ultrasound coupled with intravenous administration of microbubbles (FUS) has been proven as an effective technique to selectively and noninvasively open the BBB in multiple in vivo models including non-human primates (NHP). Although this technique has promising potential for clinical outpatient procedures, as well as a powerful tool in the lab, the safety and potential neurological effects of this technique need to be further investigated. This thesis focuses on validating the safety and efficacy of using the FUS technique to open the BBB in NHP as well as the ability of the technique to facility drug delivery. First, a longitudinal study of repeatedly applying the FUS technique targeting the basal ganglia region in four NHP was conducted to determine any potential long-term adverse side effects over a duration of 4-20 months. The safety of the technique was evaluated using both MRI as well as behavioral testing. Results demonstrated that repeated application of the FUS technique to the basal ganglia in NHP did not generate permanent side effects, nor did it induce a permanent opening of the BBB in the targeted region. The second study investigated the potential of the FUS technique as a method to deliver drugs, such as a low dose of haloperidol, to the basal ganglia in NHP and mice to elicit pharmacodynamical effects on responses to behavioral tasks. After opening the BBB in the basal ganglia of mice and NHP, a low dose of haloperidol was successfully delivered generating significant changes in their baseline motor responses to behavioral tasks. Domperidone was also successfully delivered to the caudate of NHP after opening the BBB and induced transient hemilateral neglect. In the final section of this thesis, the safety and efficacy of the FUS technique was evaluated in fully alert NHP. The FUS technique was successful in generating BBB opening volumes larger on average to that of the BBB opening volumes in anesthetized experiments. Safety results through MRI verification as well as behavioral testing during application of the technique demonstrated that the FUS technique did not generate adverse neurological effects. Conversely, the FUS technique was found to induce slight positive effects on the response of the NHP to the behavioral task. Collectively, the work presented in this thesis demonstrates the safety and effectiveness of the FUS technique to open the BBB and deliver neuroactive drugs in the NHP.

Damage to articular cartilage, whether through degeneration (i.e. osteoarthritis) or acute injury, is particularly debilitating due to the tissue's limited regenerative capacity. These impairments are common: nearly 27 million Americans suffer from osteoarthritis and 36% of athletes suffer from traumatic cartilage defects. Allografts are the preferred treatment for large cartilage defects, but demand for these tissues outweighs their supply. To generate additional replacement tissues, tissue engineering strategies have been studied as a cell-based alternative therapy. Our laboratory has had great success repeatedly achieving native or near-native mechanical and biochemical properties in grafts engineered from chondrocyte-seeded agarose hydrogels. The most common iteration of this technique yields a disk of ~4 mm diameter and ~2.3 mm thickness. However, much work is still needed to increase the potential for clinical translation of this product. Our laboratory operates under the premise that in vivo success is predicated on replicating native graft properties in vitro. Compared to these engineered grafts, native grafts are larger in size. They consist of cartilage, which has properties varying in a depth-specific manner, anchored to a porous subchondral bone base. They are able to be stored between harvest and transplantation. This dissertation presents strategies to address a subset of the remaining challenges of reproducing these aspects in engineered grafts. First, graft macrostructure was addressed by incorporating a porous base to generate biomimetic osteochondral grafts. Previous studies have shown advantages to using porous metals as the bony base. Likewise, we confirmed that osteochondral constructs can be cultured to robust tissue properties using porous titanium bases. The titanium manufacturing method, selective laser melting, offers precise control, allowing for tailoring of base shape and pore geometry for optimal cartilage growth and osteointegration. In addition to viability studies, we investigated the influence of the porous base on the measured mechanical properties of the construct's gel region. Through measurements and correlation analysis, we linked a decrease in measured mechanical properties to pore area. We promote characterization of such parameters as an important consideration for the generation of functional grafts using any porous base. Next, we investigated a high intensity focused ultrasound (HIFU) denaturation of gel-incorporated albumin as a strategy for inducing local tissue property changes in constructs during in vitro growth. HIFU is a low cost, non-contact, non-invasive ultrasound modality that is used clinically and in the laboratory for such applications as ablation of uterine fibroids and soft tissue tumors. Denaturing such proteins has been shown to increase local stiffness. We displayed the ability incorporate albumin into tissue engineering relevant hydrogels, alter transport properties, and visualize treatment with its denaturation. HIFU treatment is aided by the porous metal base, allowing for augmented heating. Though heating cartilage is used in the clinic, it is associated with cell death. We investigated this effect, finding that the associated loss of viability remains localized to the treatment zone over time. This promotes the option of balancing desired changes in tissue properties against the concomitant cell viability loss. In order to match clinically utilized allografts, engineered constructs must be scaled up in size. This process is limited by diffusional transport of nutrients and other chemical factors, leading to preferential extracellular matrix deposition in the construct periphery. Many methods are being investigated for overcoming this limitation in fixed-size constructs. In this chapter, we investigated a novel strategy in which small constructs are cultured for future assembly. This modular assembly offers coverage of variable sized defects with more consistent growth with more uniform distribution of biochemical constituents than large constructs cultured on their own. Physiologic failure testing showed that integration of these tissues may be strengthened by increased subunit strength or assembled culture. It is expected that bioadhesive caulking and/or the incorporation of osteochondral bases would further increase integration of the assembled large graft. Finally, we sought to provide a preservation/storage protocol for engineered cartilage constructs. Such a technique is critical for clinical translation, providing the engineered graft with a “shelf-life.” We adopted and evaluated the Missouri Osteochondral Allograft Preservation System (MOPS), which had been shown to maintain cell viability in native grafts for at least 63 days at room temperature without serum or growth factors. Within the current clinical of 28 days, MOPS maintained chondrocyte viability and 75% of the pre-preservation Young's modulus without significant decline in biochemical content, however it did not extend the clinical window as it had with native grafts. Refrigeration with MOPS did not show any benefit at day 28, but proved better with longer preservation times. These results are the first evaluating engineered cartilage storage. Further optimization is necessary to extend storage tissue property maintenance in storage. Overall, this dissertation presents four strategies for increasing the translation potential of engineered articular cartilage grafts by better matching the clinically utilized native allograft system. Combining these techniques, one could ideally engineer small, interlocking ostechondral constructs with HIFU modified interface properties, which could be stored from maturity to implantation. Future optimization is required to better understand and utilize these methods to engineer fully functional, clinically relevant grafts.

Breast cancer is the most common cancer and the second leading cause of cancer death among women. About 1 in 8 U.S. women (about 12%) will develop invasive breast cancer over the course of their lifetime. Existing methods of early detection of breast cancer include mammography and palpation, either by patient self-examination or clinical breast exam. Palpation is the manual detection of differences in tissue stiffness between breast tumors and normal breast tissue. The success of palpation relies on the fact that the stiffness of breast tumors is often an order of magnitude greater than that of normal breast tissue, i.e., breast lesions feel ''hard'' or ''lumpy'' as compared to normal breast tissue. A mammogram is an x-ray that allows a qualified specialist to examine the breast tissue for any suspicious areas. Mammography is less likely to reveal breast tumors in women younger than 50 years with denser breast than in older women. When a suspicious site is detected in the breast through a breast self-exam or on a screening mammogram, the doctor may request an ultrasound of the breast tissue. A breast ultrasound can provide evidence about whether the lump is a solid mass, a cyst filled with fluid, or a combination of the two. An invasive needle biopsy is the only diagnostic procedure that can definitely determine if the suspicious area is cancerous. In the clinic, 80% of women who have a breast biopsy do not have breast cancer. Most women with breast cancer diagnosed will have some type of surgery to remove the tumor. Depending on the type of breast cancer and how advanced it is, the patient might need other types of treatment as well, such as chemotherapy and radiation therapy. Image-guided minimally-invasive treatment of localized breast tumor as an alternative to traditional breast surgery, such as high intensity focused ultrasound (HIFU) treatment, has become a subject of intensive research. HIFU applies extreme high temperatures to induce irreversible cell injury, tumor apoptosis and coagulative necrosis. Compared with conventional surgical procedures the main advantages of HIFU ablation lie in the fact that it is non-invasive, less scarring and less painful, allowing for shorter recovery time. HIFU can be guided by MRI (MRgFUS) or by conventional diagnostic ultrasound (USgFUS). Worldwide, thousands of patients with uterine fibroids, liver cancer, breast cancer, pancreatic cancer, bone tumors, and renal cancer have been treated by USgFUS. In this dissertation, the objective is to develop an integrated Harmonic Motion Imaging guided Focused Ultrasound (HMIgFUS) system as a clinical monitoring technique for breast HIFU with the added capability of detecting tumors for treatment planning, evaluation of tissue stiffness changes during HIFU ablation for treatment monitoring in real time, and assessment of thermal lesion sizes after treatment evaluation. A new HIFU treatment planning method was described that used oscillatory radiation force induced displacement amplitude variations to detect the HIFU focal spot before lesioning. Using this method, we were able to visualize the HMIgFUS focal region at variable depths. By comparing the estimated displacement profiles with lesion locations in pathology, we demonstrated the feasibility of using this HMI-based technique to localize the HIFU focal spot and predict lesion location during the planning phase. For HIFU monitoring, a HIFU lesion detection and ablation monitoring method was first developed using oscillatory radiation force induced displacement amplitude variations in real time. Using this method, the HMIgFUS focal region and lesion formation were visualized in real time at a feedback rate of 2.4 Hz. By comparing the estimated lesion size against gross pathology, the feasibility of using HMIgFUS to monitor treatment and lesion formation without interruption is demonstrated. In order to reduce the imaging time, it is shown in this dissertation that using the steered FUS beam, HMI can be used to image a 2.3 times larger ROI without requiring physical movement of the transducer. Using steering for HMI can be used to shorten the total imaging duration without requiring physical movement of the transducer. For the application of breast tumor, HMI and HMIgFUS were optimized and applied to ex vivo breast tissue. The results showed that HMI is experimentally capable of mapping and differentiating stiffness in normal and abnormal breast tissues. HMIgFUS can also successfully generate thermal lesions on normal and pathological breast tissues. HMI has also been applied to post-surgical breast mastectomy specimens to mimic the in vivo environment. In the end, the first HMI clinical system has been built with added capability of GUP-based parallel beamforming. A clinical trial has been approved at Columbia University to image breast tumor on patient. The HMI clinical system has shown to be able to map fibroadenoma mass on two patients with valid HMI displacement. The study in this dissertation may yield an early-detection technique for breast cancer without any age discrimination and thus, increase the survival rate.

Real-time acousto-optic (AO) sensing - a dual-wave modality that combines ultrasound with diffuse light to probe the optical properties of turbid media - has been demonstrated to non-invasively detect changes in ex vivo tissue optical properties during high-intensity focused ultrasound (HIFU) exposure. The AO signal indicates the onset of lesion formation and predicts resulting lesion volumes. Although proof-of-concept experiments have been successful, many of the underlying parameters and mechanisms affecting thermally induced optical property changes and the AO detectability of HIFU lesion formation are not well understood. In thesis, a numerical simulation was developed to model the AO sensing process and capture the relevant acoustic, thermal, and optical transport processes. The simulation required data that described how optical properties changed with heating. Experiments were carried out where excised chicken breast was exposed to thermal bath heating and changes in the optical absorption and scattering spectra (500 nm - 1100 nm) were measured using a scanning spectrophotometer and an integrating sphere assembly. Results showed that the standard thermal dose model currently used for guiding HIFU treatments needs to be adjusted to describe thermally induced optical property changes. To model the entire AO process, coupled models were used for ultrasound propagation, tissue heating, and diffusive light transport. The angular spectrum method was used to model the acoustic field from the HIFU source. Spatial-temporal temperature elevations induced by the absorption of ultrasound were modeled using a finite-difference time-domain solution to the Pennes bioheat equation. The thermal dose model was then used to determine optical properties based on the temperature history. The diffuse optical field in the tissue was then calculated using a GPU-accelerated Monte Carlo algorithm, which accounted for light-sound interactions and AO signal detection. The simulation was used to determine the optimal design for an AO guided HIFU system by evaluating the robustness of the systems signal to changes in tissue thickness, lesion optical contrast, and lesion location. It was determined that AO sensing is a clinically viable technique for guiding the ablation of large volumes and that real-time sensing may be feasible in the breast and prostate.

High intensity focused ultrasound (HIFU) provides a wide range of noninvasive therapies ranging from drug delivery to the destruction of kidney stones. In particular, thermal ablation by HIFU presents an effective noninvasive method for the treatment of deep seated solid tumours. HIFU’s further uptake is limited by a need for improved treatment planning and monitoring. Two nonlinear acoustic phenomena that play key roles in HIFU treatment: finite amplitude effects that lead to the generation of harmonics and steepening of wavefronts, and acoustic cavitation. The former must be taken into careful consideration for treatment planning purposes, while the latter has the potential to provide fast, real-time, cost effective treatment monitoring. The first half of this thesis provides new measurements for the nonlinear acoustic properties of tissue, assesses the validity of two common modelling techniques for simulating HIFU fields. The second half develops a new method for combining passive acoustic mapping- an ultrasound monitoring technique- with MR thermometry, to assess estimates of cavitation enhanced heating derived from passive acoustic maps. In the first results chapter B/A was measured in ex-vivo bovine liver, over a heating/ cooling cycle replicating temperatures reached during HIFU ablation, adapting a finite amplitude insertion technique (FAIS), which also allowed for measurement of sound-speed and attenuation. The method measures the nonlinear progression of a plane-wave through liver and B/A was chosen so that numerical simulations matched measured waveforms. Results showed that attenuation initially decreased with heating then increased after denaturation, sound-speed initially increased with temperature and then decreased, and B/A showed an increase with temperature but no significant post-heating change. These data disagree with other reports that show a significant change and suggest that any nonlinear enhancement in the received ultrasound signal post-treatment is likely due to acoustic cavitation rather than changes in tissue nonlinearity. In the second results chapter two common methods of modelling HIFU fields were compared with hydrophone measurements of nonlinear HIFU fields at a range of frequencies and pressures. The two methods usedwere the KZK equation and the commercial package PZFlex. The KZK equation has become the standard method for modelling focused fields, while the validity of PZFlex for modelling these types of transducers is unclear. The results show that the KZK equation is able to match hydrophone measurements, but that PZFlex underestimates the magnitude of the harmonics. Higher order harmonics in PZFlex are not the correct shape, and do not peak around the focus. PZFlex performs worse at higher pressures and frequencies, and should be used with caution. In the final two chapters a system for estimating cavitation-enhanced heating from acoustic maps is developed and benchmarked against magnetic resonance thermometry methods. The first chapter shows that the ultrasound and MR monitoring systems are compatible, and registers the two imaging systems. The HIFUfocus is clearly visible in passive maps acquired in the absence of cavitation and these coincide with the centre of heating in MR temperature images. When cavitation occurs, it coincides spatially and temporally with the appearance of a clear spike in temperature, especially when the passive maps are processed using the Robust Capon Beamformer algorithm. The final chapter shows how passive maps can be converted into thermal heating inputs, and used to estimate cavitation-enhanced temperature increases. These estimates have the potential to closely match maximum temperature rise, and estimated thermal dose after the estimated temperature rise is spatially averaged. However, themethod is not always successful. This is partly due to uncertainties in MR thermometry estimates, partly due to uncertainties in the acoustic properties of tissue.

Sabe-se que o aumento na temperatura do tecido tumoral pode aumentar a eficiência de técnicas convencionais de combate ao câncer (radioterapia e quimioterapia). Além disso, a variação de temperatura em tumores pode ser uma forma de tratamento alternativo à cirurgia, feito por meio do fornecimento de calor direcionado às células cancerosas e preservando o tecido sadio. Para maior eficácia e segurança no emprego de técnicas que utilizam fornecimento de calor ao tecido biológico, é necessário o monitoramento da temperatura tecidual para garantir que a morte celular por ablação térmica seja limitada ao tecido alvo, minimizando os danos aos tecidos adjacentes. A imagem fotoacústica é uma técnica baseada no efeito fotoacústico, o qual consiste na absorção de radiação eletromagnética pelo tecido e, devido à expansão termoelástica, na geração de ondas acústicas. A amplitude da onda de pressão gerada pelo efeito fotoacústico possui dependência com a temperatura do meio pelo parâmetro de Grueneisen, que depende das propriedades mecânicas do material. Portanto, mudanças na amplitude do sinal fotoacústico carregam informações a respeito da variação na temperatura do material. Neste trabalho, a dependência da amplitude do sinal fotoacústico com a temperatura foi estudada em um material simulador de tecido biológico (phantom) em condições similares a de tratamentos por hipertermia Nesse caso, imagens fotoacústicas foram adquiridas para cada grau de temperatura em uma faixa de 36 até 41 ºC durante o procedimento de aquecimento por banho térmico. Mudanças na amplitude e fase do sinal fotoacústico foram avaliadas através da aplicação de algoritmos de speckle tracking. Para estimar a variação na amplitude do sinal também foram utilizados e avaliados diferentes métodos de comparação. Os resultados são apresentados por imagens fotoacústicas termais produzidas pela aplicação de um fator de calibração aos mapas de variação relativa da amplitude do sinal em função da temperatura do meio. Finalmente, avaliamos um experimento de hipertemia por ultrassom focalizado de alta intensidade (High Intensity Therapeutic Ultrasound - HITU) em uma amostra de músculo suíno. Nesse caso foram geradas imagens termais fotoacústicas e imagens termais produzidas pela mudança de fase do sinal pulso-eco de ultrassom. Os resultados sugerem uma maneira não invasiva de calcular a distribuição da variação de temperatura do meio que pode ser aplicada para monitoramento durante tratamentos que utilizam o fornecimento de calor ao tecido biológico. / Several studies have shown that elevating the temperature of tumoral tissue improves standard cancer treatments success rate (radiotherapy and chemotherapy). This procedure can also be a therapy to cancer by delivering heat and killing cancer cells while healthy tissues are preserved. For improved efficiency and security in heat applications, it is important to monitor tissue temperature during treatments. Photoacoustic (PA) pressure wave amplitude has a temperature dependence given by the sample mechanical properties (Gruenesein parameter). These changes in photoacoustic signal amplitude carry information about temperature variation in tissue. Therefore, PA has been proposed as an imaging technique to monitor temperature during hyperthermia. In this study, PA images were acquired for temperatures ranging from 36ºC to 41ºC using a tissue-mimicking phantom immersed in a temperature controlled thermal bath. Relative amplitude variation was calculated using speckle tracking algorithms using four different methods to estimate these variations in PA signal amplitude. The results are presented as PA-based thermal images, generated using a calibration factor to the percentage variations in the amplitude maps. Finally, PA-based and ultrasound-based thermal images were acquired during heating by high intensity focused ultrasound (High Intensity Therapeutic Ultrasound - HITU) in a porcine muscle. The results suggest a non-invasive way to monitor temperature during hyperthermia procedures.

No description available.

Recent evidence appears to indicate that neurons, responsible for our perception of the world around us, are not only electrically excitable, but may have mechanical triggers as well. This is well supported through the growing number of observations of focused ultrasound (FUS) perturbations of the neurons located in our central nervous system (CNS). However, while the CNS is largely responsible for turning electrical signals from the periphery into thoughts and understanding, less is known about the effect of which FUS has upon the peripheral signals themselves: our peripheral nervous system (PNS). Given the non-invasive nature of FUS - were it be discovered to influence neuronal signaling, FUS would become a powerful tool for therapy and medicine, especially in conditions involving pain. Thus, we ponder the question, "How can FUS modulate nerve activity and furthermore, what are the interactions on pain signaling?" In this dissertation, a road-map is described for translating insights acquired through pre-clinical study of ultrasound PNS stimulation to clinical investigation on neuropathic pain modulation in humans. More specifically, methods and tools to study excitation of the sciatic nerve bundle and the dorsal root ganglia (DRG) were built and optimized in rodent models. In turn, these methods and findings enabled investigation into pain signaling and translation to human studies. Finally, FUS was shown to mitigate pain sensations in human patients with neuropathic pain. First, using a newly developed in vivo nerve displacement imaging technique, mechanical deformations of the nerve from FUS stimulation were noninvasively mapped in a two-dimensional plane centered at the sciatic nerve. Nerve displacements were positively correlated with downstream compound muscle activation from FUS sciatic nerve stimulation. Furthermore, by focusing ultrasound waves to the DRGs directly in an ex vivo preparation, additional parameters were identified to modulate spike transmission, effectively regulating high frequency signaling. Next, we investigated the feasibility translating FUS nerve stimulation to clinical studies. We first looked at effects on upstream cortical activity and pain signaling from somatosensory stimuli using high-frequency functional ultrasound (fUS) imaging. FUS was shown to both stimulate somatosensation and suppress pain signaling in the cortex. Secondly, nerve displacement imaging was scaled-up for human investigation, essential for in-procedure localization and stimulation of the targeted nerve bundle. Using a combination of imaging and therapeutic excitation, simultaneous nerve targeting, stimulation, and monitoring was established at pressures required for stimulation. Lastly, clinical feasibility was investigated using previously optimized FUS pulse schemes and scaled-up neuromodulation technologies. Specifically, we applied simultaneous FUS to the median nerve and thermal stimulation to the corresponding dermatome in healthy human subjects. Furthermore, patients with robust and repeatable mechanically-assessed neuropathic pain were similarly stimulated with FUS to assess pain suppression. Based on the findings presented herein, noninvasive FUS peripheral stimulation has the potential for radically shifting the traditional pharmaceutical paradigms in chronic and acute pain treatment by altering signals before being processed in the spinal cord and ultimately the brain. The studies outlined herein serve to elucidate mechanisms of FUS in the PNS, as well as provide the starting foundations for further development of FUS as an effective pain treatment.

In addition to protecting the brain from circulating pathogens and neurotoxins, the blood-brain barrier (BBB) limits both the delivery of drugs to the brain and the migration of neurological disease biomarkers from the brain into the blood. Focused-ultrasound blood-brain barrier opening (FUS-BBBO) addresses both of these transport limitations by transiently and noninvasively opening the BBB. Although originally designed as a drug delivery method, FUS-BBBO has also been shown to be an effective neuroimmunotherapy and method of improving liquid biopsy specificity for neurological disease. Prior to the work presented herein, the mechanism of FUS-BBBO neuroimmunotherapy remained poorly characterized and FUS-BBBO liquid biopsy remained poorly optimized. Initially, we present the temporal response of brain macrophages to FUS-BBBO. Due totheir role as the main phagocyte in the brain and the well-documented association between their dysfunction and neurodegenerative disease progression, we hypothesized that FUS-BBBO affects brain macrophage population composition and phenotype. Utilizing temporal single-cell RNA sequencing, we establish that treatment remodels the immune landscape via a number of processes including microglia proliferation, disease-associated microglia population size increase, and central-nervous-system associated macrophage recruitment. To further elucidate the functional role of the brain macrophage response to FUS-BBBO, we find that their depletion is associated with significantly decelerated BBB restoration. Secondly, we compare FUS-BBBO with two other methods of focused ultrasound neuroimmunotherapy, focused ultrasound neuromodulation (FUS-N) and focused ultrasound with microbubbles without BBBO (FUS+MB). FUS-N utilizes FUS parameters that alter neuronal connectivity via a combination of mechanosensitive receptor interactions and transient hypothermia without the injection of microbubbles (MB). FUS+MB is the combination of MB and FUS below the pressure threshold for BBBO (FUS+MB). FUS+MB has been shown to trigger morphological activation of brain macrophages and has proven efficacious as a method of immunotherapy within the peripheral nervous system. Due to the findings of brain macrophage modulation in response to FUS-BBBO, we compare brain macrophage modulation between all three paradigms both in the presence and absence of Alzheimer’s Disease (AD) pathology. We identify FUS-BBBO as the paradigm which maximizes brain macrophage modulation including an increase in the population of neuroprotective, disease-associated microglia and direct correlation between FUS cavitation dose and brain macrophage phagocytosis. Next, we combine spatial and single-cell transcriptomics with immunohistochemical validation to characterize the effect of FUS-BBBO on brain macrophage distribution in both wild-type and Alzheimer’s disease animals. Given their relevance within neurodegeneration and perturbation response, we emphasize the distribution of three brain macrophage populations - disease- and interferon-associated microglia and central-nervous-system-associated macrophages. We find a genotype-specific redistribution of each population, with an overall trend towards increased interaction with the brain-cerebrospinal fluid barrier after FUS-BBBO, an effect that is found to be more pronounced in the presence of disease pathology. Finally, we investigate the role of extracellular vesicles (EVs) in both the mechanism ofFUS-BBBO neuroimmunotherapy and as a method of improving FUS-BBBO liquid biopsy. EVs are lipid vesicles that are responsible for the transport and exchange of diverse cargo between cells and have been reported to modulate the immune system. Isolation of EVs has emerged as a method of improving biomarker detection. Prior to this study, the effect of FUS-BBBO neuroimmunotherapy on EV concentration and content remained unexplored. We investigate the concentration and content of isolated EVs from the serum of mice and Alzheimer’s Disease patients prior to and after treatment with FUS-BBBO. We illustrate a 100% increase in EV concentration one hour after treatment in both mice and patients. Furthermore, we illustrate an increase in murine EV RNA that is associated with the previously reported neuroimmunotherapeutic responses to FUS-BBBO including synaptic remodeling and neurogenesis. Finally, we illustrate an increase in AD biomarker concentration within the patient EVs three days after treatment that is proportional to the volume of blood-brain barrier opening. Overall, we establish that FUS-BBBO drug-free neuroimmunotherapy triggers complex brain macrophage modulation in a manner incomparable by other FUS neuroimmunotherapy paradigms. Furthermore, we illustrate the effect of FUS-BBBO on EV concentration and content in both preclinical and clinical experiments, indicating the role of EVs in FUS-BBBO neuroimmunotherapy and their utility as a method of improving liquid biopsy specificity. The results presented herein support the potential of FUS-BBBO as both a method of neuroimmunotherapy and a method of amplifying liquid biopsy specificity in Alzheimer’s Disease.

Focused ultrasound (FUS) can modulate activity in the central nervous system of animals, however the mechanism of action is not yet fully understood. FUS is a promising technique for clinical use in treating both physiological and psychological pathology of the nervous system. FUS can noninvasively penetrate the skull deep into the brain and modulate brain targets with millimeter-scale resolution. FUS is less invasive than deep brain stimulation (DBS) and can target deeper structures with greater resolution than transcranial magnetic stimulation (TMS). Functional ultrasound imaging (fUSI) is an emerging modality for monitoring stimulus-evoked brain activity. However, the thick skull of large animals poses a significant obstacle for the noninvasive translation of the technique to nonhuman primates and humans. In this dissertation, FUS is performed in mice and nonhuman primates and an fUSI technique is developed for transcranially imaging FUS-evoked responses in both species. The first aim of this dissertation established a procedure for performing high-resolution FUS in mice in vivo. FUS-evoked motor responses were evaluated using four-limb electromyography (EMG). A detailed quantitative analysis of several EMG characteristics demonstrated that observed motor responses exhibited brain target-specific differences. FUS in the brain was also shown to modulate cardiorespiratory activity. However, simulations conceded that intracranial reverberations may activate brain structures outside acoustic foci, suggesting that direct detection of brain activity is preferable to responses like EMG and cardiorespiratory activity. The second aim of this dissertation developed an fUSI system for monitoring FUS-evoked responses in mice in vivo. fUSI was validated using electrical peripheral nerve stimulation to elicit somatosensory-evoked responses, a well-characterized approach in established techniques like functional magnetic resonance imaging (fMRI). fUSI was later integrated into an ultrasoundbased optogenetic stimulation procedure. Lastly, a dual FUS-fUSI transducer system for performing neuromodulation and functional activity monitoring was developed and successfully demonstrated in mice in vivo. The final aim of this dissertation was to adapt the FUS-fUSI procedure developed in mice for use in nonhuman primates. Two approaches were developed and tested in vivo. The first approach employed a low-frequency ultrasound array for both neuromodulation and activity monitoring. The second approach implemented a dual FUS-fUSI transducer system similar to that used in mice. Preliminary evidence indicated that the adapted dual transducer system can successfully perform fully noninvasive neuromodulation and functional activity monitoring transcranially in nonhuman primates in vivo. The findings presented in this dissertation provide a framework for performing fully noninvasive ultrasound-mediated neuromodulation and functional activity monitoring in non human primates and describes a road map for further translating the technique for clinical use in human subjects. A fully noninvasive FUS-fUSI technique can provide an invaluable tool for clinicians to treat diseases of the nervous system not indicated for invasive procedures, opening the door to a wide range of therapeutic applications.