Studies of the Effects of Therapeutic Ultrasound Using Various Imaging Modalities
Open AccessOur lab has recently shown that ultrasound can induce a reversible release of insulin from cultured rat beta cells and that this release is calcium dependent and is not destructive to the cells. These promising results are a large step toward new possible methods for treatment of type 2 diabetes and other secretory diseases of the endocrine system. These novel treatments are much needed given the difficult nature of pharmacological management of the type 2 diabetes. Another relatively unexplored area of therapeutic ultrasound (TUS) is its effects on the heart specifically in cardiomyocyte cultures. This is highly relevant to the field of tissue engineering where possible reparative cardiac therapies would require cardiomyocytes expressing a mature phenotype. To mature, cardiomyocytes require mechanical stimulation, something ultrasound can provide. Ultrasound may also provide a way to constantly and non-invasively monitor cell cultures with the use of spectral ultrasound (SUS) imaging. Not much work has been done on SUS’s ability to monitor structures on a small scale such as cell culture, or its ability to monitor changes on very short time scales. In the work presented in this dissertation, we quantified and assessed dynamical metabolic changes in an in situ pancreatic slice model evoked by ultrasound application. After plating, pancreas slices were imaged using a confocal microscope at 488 nm and 633 nm to image lipoamide dehydrogenase (Lip-DH) autofluorescence and a far red fluorescence respectively. Ultrasound was applied at intensities of 0.5 and 1 W/cm2 at both 800 kHz and 1 MHz. Additionally 800 kHz ultrasound at 1 W/cm2 was applied in a pulsing scheme. Far red fluorescence revealed significant differences between all experimental groups and control in the pancreatic islets (p<0.05) and between all ultrasound experimental groups and control (p<0.05) in pancreatic exocrine tissues. However, this difference in response between control and glucose did not exist in the exocrine tissue. We also observed that glucose produces a significantly increased metabolic response in islet tissue compared to exocrine tissue (p<0.05). Pulsed ultrasound appeared to increase metabolic activity in the pancreatic slice in a more consistent manner. Our results indicate that TUS may have a stimulatory metabolic effect on the pancreatic islets similar to glucose. Further, in this dissertation we explore the effects of a set of TUS parameters on human induced pluripotent stem cell cardiomyocytes (iPS-CMs). Ultrasound was applied at 600 kHz at 1, 3.4 and 6 W/cm2 for a continuous one second pulse. iPS-CMs were imaged with calcium fluorescence microscopy while TUS was being applied. Measures of the instantaneous beat frequency, repolarization rate and calcium spike amplitude were calculated from the fluorescence data. TUS at 600 kHz 1 W/cm2 and 6 W/cm2 had significant effects in the shortening of both the repolarization rate as well as the instantaneous beat rate (p<0.05). Further, TUS at 3.4 W/cm2 and 6 W/cm2 had significant effects in the shortening of the calcium spike amplitude. Three SUS measures and one gray level measure were calculated from ultrasound reflections of the beating iPS-CM monolayer while they were simultaneously being imaged with the Vevo 3100 ultrasound imager and calcium labeled confocal microscopy. The gray level measure performed the best out of all tested measures, however it was not reliable enough to produce a consistent measure of the beat rate of the cell. Finally SUS measures were captured in three modes, (1) the Vevo 3100 and an MX250 transducer, (2) an MX550D transducer and (3) an Olympus 50 MHz unfocused transducer. Ultrasound radio frequency (RF) data was captured from the iPS-CM monolayers before and after TUS. A center of mass measure calculated from the wavelet transform scalogram of the time averaged RF data revealed that a change in response to TUS was detectable by the Olympus 50 MHz transducer at 1 W/cm2 with significance (p<0.05). Further this measure was able to detect a shift in the time information at 6 W/cm2 with the Vevo 3100 MX250 transducer and a shift in the frequency information at 6 W/cm2 detected by the MX550D transducer (p<0.05). Overall, SUS showed promise as a method for constant monitoring of tissue cultures.
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