Investigations into the safety of diagnostic ultrasound and mechanisms of therapeutic ultrasound have provided evidence of a number of cellular responses to ultrasound. These studies have mainly concentrated on cells in culture, while work on intact tissue employed mainly kHz ultrasound fields, although diagnostic and many therapeutic procedures are performed using MHz ultrasound. Vascular tissue is known to respond to a variety of physical and chemical signals, and so arteries were used as a model system in this thesis to study the effects of MHz ultrasound in vitro. Rings of equine carotid and lateral cecal mesenteric artery exhibited reversible, repeatable contraction on exposure to both pulsed and continuous wave 3.2 MHz ultrasound at acoustic powers up to 145 mW. Wall stress increased by up to 1.5% in carotid arteries and up to 2% in mesenteric arteries during exposure, and returned to basal levels after approximately 10 minutes. Contraction was endothelium independent, and was not affected by changes in the pulsing regime. The magnitude of contraction was dependent on the acoustic power, and the change in wall stress increased with increasing acoustic power in a linear fashion. The acoustic power dependence suggested the response was thermally mediated and this was confirmed by investigation of the response of arteries to non ultrasound generated heating, which also induced contraction. The effects of ultrasound and heating were also investigated in 1st order branches of the lateral cecal artery, as a model of a small artery. No response was observed in either case. In order to determine the cellular basis of the response of carotid and mesenteric arteries, the involvement of potassium ion channels in the response was investigated using a potassium channel blocker. The response of arteries to ultrasound was increased by inhibition of inward-rectifier potassium channels, which would otherwise help to return the cell membrane potential to the normal level. The change in wall stress was increased by 42% on average, confirming the involvement of these channels in the response. Contraction of arteries is mediated by an increase in intracellular calcium. The ion channel activity during non ultrasound generated heating was examined further by observation of intracellular calcium concentration using a fluorescent calcium sensitive dye. Increases in intracellular calcium were observed in carotid and large mesenteric arteries, which confirmed the thermal influence on ion channel function in these vessels. No such effect was observed in the smaller vessels.

The inability to transport molecules efficiently and easily into cells and across tissues is one of the major limitations of developing drug delivery systems. A novel approach to overcoming this problem could be the use of low-frequency ultrasound to make cell membranes and tissues more permeable. Previous studies show that normally impermeant molecules can be transported into cells exposed to ultrasound; however, the mechanism by which this occurs is not well understood. Our hypothesis is that low frequency ultrasound can reversibly disrupt membrane structure, thus allowing diffusion-driven intracellular delivery of molecules through a breach in the cell membrane. The effects of ultrasound are not limited to uptake of molecules; there can also be significant loss of cell viability after sonication. Therefore, the focus of this work is to determine the mechanisms by which molecular uptake and cell death occur from ultrasound exposure. The long-term goal of this work is to increase the number of viable cells that experience uptake by controlling the effects that cause cell death. Our data have show that large molecules (r ≤ 28 nm) can be taken into cells after exposure to 24 kHz (10% duty cycle for 2 s of exposure time at 0.1 pulse length over a range of pressures) ultrasound and that uptake of these molecules can occur even after sonication ended. In experiments developed to isolate the mechanism(s) of uptake, DU145 prostate cancer cells depleted of ATP energy and intracellular calcium showed no uptake of calcein, a small fluorescent molecule (MW = 623 Da), nor did sonicated lipid bilayers (red blood cell ghosts), suggesting that uptake is calcium mediated and requires active mechanisms in viable cells. Multiple types of microscopy, including electron and laser scanning confocal, showed evidence of large plasma membrane disruptions which support the hypothesis that transport of molecules into cells occurs through repairing wounds. Microscopy studies also indicated that much if the sonication-mediated death can occur by instantaneous cellular lysing and rapid cell death (within minutes post-exposure) due to wound-instigated necrosis; in addition, characteristics of rapidly induced controlled death modes were seen and found to be non-caspase-mediated within an hour after sonication ended.

No description available.

Tendon injuries are prevalent, debilitating and difficult to treat. Common interventions such as anti-inflammatory medication, growth factor injections and surgery are associated with short-term efficacy and long rehabilitation periods. Tendons possess an incomplete healing response which is reparative (scar-mediated) rather than regenerative, resulting in a 'healed' tissue that is mechanically inferior to the native tendon. While it is widely accepted that mechanical-loading based treatments offer long-term symptomatic resolution and improved functionality, the exact mechanisms of action of such mechanotransduction-based healing cascades remain unclear. Nevertheless, there is significant motivation for the development of non-invasive and efficient rehabilitative treatments that mechanically stimulate the injured tendons to achieve functional healing responses. Focused Ultrasound (FUS) methods are an attractive treatment option as they are non-invasive, utilize higher intensities for shorter durations and are targeted to a very specific treatment volume, hence inducing significant bio-effects in the tissue without affecting surrounding structures. Herein, we present a body of work that includes the development of FUS pulsing to precisely target murine Achilles tendons and emphasize distinct bioeffects (thermal-dominant and mechanical-dominant). We investigated the feasibility of applying FUS pulsing to murine Achilles tendons ex vivo and in vivo and demonstrated that FUS can be safely applied without any deleterious effects in the tendons and surrounding tissues. The animals showed no symptoms of distress after multi-session treatments. Overall, results suggest that tendon material properties are not adversely altered by FUS pulsing. Histological analyses showed mild matrix disorganization, suggesting the need for slight modifications in the ultrasound pulsing parameters and treatment durations. When applied to injured tendons, mechanical dominant schemes seemed to drive larger improvements in material properties compared to thermal-dominant pulsing, confirming our original hypothesis that mechanical stimulation may play a bigger role in tendon healing compared to purely thermal-dominant stimulation. Additionally, feasibility of histotripsy ablation in murine Achilles tendons was successfully investigated ex vivo and in vivo and experimentation to further optimize these methods are ongoing. Such (non-thermal) ablative paradigms will be extremely useful when conservative treatment options are unavailable and debridement of scar tissue is warranted to interrupt the degenerative process and stimulate healing. Finally, a pilot investigation into FUS-induced strains was performed to guide our parameter selection process and deliver controlled strains to achieve healing responses (similar to current clinical rehabilitation protocols). We were able confirm that strains between 1% and 6% (or higher) can be induced by manipulating ultrasound treatment parameters. Overall, or results reiterate the potential of FUS in eliciting the desired bioeffects and thus achieve healing in tendons and provide a snapshot of the expected effects of using such pulsing methods to treat tendon injuries. / Doctor of Philosophy / Tendons are tissues that connect muscles to bones, and are unfortunately prone to injuries. Such injuries are prevalent and difficult to treat. Effective treatment options remain limited, as common methods such as surgery, anti-inflammatory medications and corticosteroid injections do not provide long-term relief. One of the few treatments that has been proven to provide symptomatic relief and improved the functionality of chronically (over a long period of time) injured tendons is physical therapy. However, researchers are still investigating the reasons for this successful healing response. Some limitations of physical therapy are long rehabilitation and recovery periods, and the need for patient compliance (i.e., performing painful exercises while already being under significant pain). In this research, we explore the use of a non-invasive modality known as ultrasound to treat tendon injuries. Ultrasound is commonly thought of as a diagnostic tool, i.e., to detect injuries in musculoskeletal medicine. It, however, is also an attractive therapeutic (treatment) modality, as sound waves can be concentrated in the required area of interest which results in different types of effects in the chosen tissue, such as heating. A huge advantage is that ultrasound is non-invasive, painless, and safe, as the energy is only applied to the chosen volume of interest and surrounding structures are unaffected. To examine the utility of therapeutic ultrasound in treating tendon injuries, we used a mouse model that has been previously used in our lab, and designed different types of ultrasound treatments that elicit two main types of effects in the tissue, namely, thermal, or heating effects and mechanical, or physical therapy-like effects. Prior to applying these treatments, we measured how much heating is produced in mouse Achilles tendons via these treatments, to establish safety. Once we identified safe thermal and mechanical treatment sets, we treated mouse Achilles tendons ex vivo, i.e., after euthanasia. We tested the mechanical properties of the treated tendons and determined that treatments do not alter the mechanical properties of tendons, which is encouraging, given that we do not want treatments to interfere with the properties of native tendons. We also examined the influence of treatments on structure of Achilles tendons after treatments and deducted that the structure was not damaged due to treatments. We followed up these studies with treatments conducted in live mice, which received four treatment sessions in one week. These studies were conducted to further determine the safety and tolerance to these procedures and also examine the healing effects of treatments in injured Achilles tendons. Results suggest that focused ultrasound treatments are safe and tolerable to mice and seem to elicit improvements in tendon properties. In other studies, we also examined a different ultrasound method named histotripsy, as a non-invasive alternative to dry needling (which is another methodology used to treat tendon injuries) and scar debridement (removal of scar tissue to stimulate a new healing response). This research establishes that therapeutic ultrasound is a novel, non-invasive alternative with good potential to treat tendon injuries. Future studies will investigate the effects of ultrasound treatments over longer durations and also aim to clarify the exact type and magnitude of physical therapy-like forces that are produced by ultrasound treatments. This understanding will enhance our treatment design process to be able to mimic clinical treatments that are known to be effective.

No description available.