Analyzing Gene Expression After Administration of Low-Intensity Therapeutic Ultrasound in Human Islet Cells.

OBJECTIVES: Diabetes mellitus is a complex heterogenous metabolic disease that significantly affects the world population. Although many treatments exist, including medications such as metformin, sulfonylureas, and glucagon-like peptide-1 (GLP) receptor agonist, there is growing interest in finding alternative methods to noninvasively treat this disease. It has been previously shown that low-intensity ultrasound stimulation of pancreatic ß-cells in mice can elicit insulin secretion as a potential treatment for this disease. This is desirable as therapeutic ultrasound has the ability to induce bioeffects while selectively focusing deep within tissues, allowing for modulation of hormone secretion in the pancreas to mitigate insufficient levels of insulin. METHODS: Exactly 800 kHz ultrasound with intensity 0.5 W/cm2 was administered 5 minutes continuously, that is, 100% duty cycle, to donor pancreatic human islets, followed by 1 hour incubation and RT-qPCR to assess the effect of ultrasound stimulation on gene expression. The genes were insulin (INS), glucagon (Glu), amylin (Amy), and binding immunoglobulin protein (BiP). Nine donor pancreatic human islets were used to assess insulin and glucagon secretion, while eight samples were used for amylin and BiP. Fold change (FC) was calculated to analyze the effect of ultrasound stimulation on the gene expression of the donor islet cells. High-glucose and thapsigargin-treated islets were utilized as positive controls. Cell viability testing was done using a Trypan Blue Exclusion Test. RESULTS: Ultrasound stimulation did not cause a statistically significant upregulation in any of the tested genes (INS FC = 1.15, P-value = .5692; Glu FC = 1.60, P-value = .2231; Amy FC, P-value = .2863; BiP FC = 2.68, P-value = .3907). CONCLUSIONS: The results of this study show that the proposed ultrasound treatment parameters do not appear to significantly affect gene expression of any gene tested.

Therapeutic Ultrasound for Enhanced Corneal Permeability to Macromolecules.

OBJECTIVES: Topically applied macromolecules have the potential to provide vision-saving treatments for many of the leading causes of blindness in the United States. The aim of this study was to determine if ultrasound can be applied to increase transcorneal drug delivery of macromolecules without dangerously overheating surrounding ocular tissues. METHODS: Dissected corneas of adult rabbits were placed in a diffusion cell between a donor compartment filled with a solution of macromolecules (40, 70 kDa, or 150 kDa) and a receiver compartment. Each cornea was exposed to the drug solution for 60 minutes, with the experimental group receiving 5 minutes of continuous ultrasound or 10 minutes of pulsed ultrasound at a 50% duty cycle (pulse repetition frequency of 500 ms on, 500 ms off) at the beginning of treatment. Unfocused circular ultrasound transducers were operated at 0.5 to 1 W/cm2 intensity and at 600 kHz frequency. RESULTS: The greatest increase in transcorneal drug delivery seen was 1.2 times (P < .05) with the application of pulsed ultrasound at 0.5 W/cm2 and 600 kHz for 10 minutes with 40 kDa macromolecules. Histological analysis revealed structural damage mostly in the corneal epithelium, with most damage occurring at the epithelial surface. CONCLUSIONS: This study suggests that ultrasound may be used for enhancing transcorneal delivery of macromolecules of lower molecular weights. Further research is needed on the long-term effects of ultrasound parameters used in this study on human ocular tissues.

Modeling of Ultrasound Stimulation of Adolescent Pancreas for Insulin Release Therapy.

OBJECTIVES: Our previous published studies have focused on safety and effectiveness of using therapeutic ultrasound (TUS) for treatment of type 2 diabetes mellitus (T2DM) in preclinical models. Here we present a set of simulation studies to explore potential ultrasound application schemes that would be feasible in a clinical setting. METHODS: Using the multiphysics modeling tool OnScale, we created two-dimensional (2D) models of the human abdomen from CT images captured from one normal weight adolescent patient, and one obese adolescent patient. Based on our previous studies, the frequency of our TUS was 1 MHz delivered from a planar unfocused transducer. We tested five different insonation angles, as well as four ultrasound intensities combined with four different duty factors and five durations of application to explore how these variables effect the peak pressure and temperature delivered to the pancreas as well as surrounding tissue in the model. RESULTS: We determined that ultrasound applied directly from the anterior of the patient abdomen at 5 W/cm2 delivered consistent acoustic pressures to the pancreas at the levels which we have previously found to be effective at inducing an insulin release from preclinical models. CONCLUSIONS: Our modeling work indicates that it may be feasible to non-invasively apply TUS in clinical treatment of T2DM.

Therapeutic Ultrasound for Topical Corneal Delivery of Macromolecules.

Purpose: The objective of this study was to utilize therapeutic ultrasound in enhancing delivery of topical macromolecules into the cornea. Methods: Rabbit corneas were dissected and placed in a diffusion cell with a small ultra-red fluorescent protein (smURFP; molecular weight of 32,000 Da) as a macromolecule solution. The corneas were treated with continuous ultrasound application at frequencies of 400 or 600 kHz and intensities of 0.8 to 1.0 W/cm2 for 5 minutes, or sham-treated. Fluorescence imaging of the cornea sections was used to observe the delivery of macromolecules into individual epithelial cells. Spectrophotometric analysis at smURFP maximal absorbance of 640 nm was done to determine the presence of macromolecules in the receiver compartment. Safety of ultrasound application was studied through histology analysis. Results: Ultrasound-treated corneas showed smURFP delivery into epithelial cells by fluorescence in the cytoplasm, whereas sham-treated corneas lacked any appreciable fluorescence in the individual cells. The sham group showed 0% of subcellular penetration, whereas the 400 kHz ultrasound-treated group and 600 kHz ultrasound-treated group showed 31% and 57% of subcellular penetration, respectively. Spectrophotometry measurements indicated negligible presence of smURFP macromolecules in the receiver compartment solution in both the sham and ultrasound treatment groups, and these macromolecules did not cross the entire depth of the cornea. Histological studies showed no significant corneal damage due to ultrasound application. Conclusions: Therapeutic ultrasound application was shown to increase the delivery of smURFP macromolecules into the cornea. Translational Relevance: Our study offers a clinical potential for a minimally invasive macromolecular treatment of corneal diseases.

Therapeutic Ultrasound Effects on Human Induced Pluripotent Stem Cell Cardiomyocytes Measured Optically and with Spectral Ultrasound.

To the best of our knowledge, therapeutic ultrasound (TUS) is thus far an unexplored means of delivering mechanical stimulation to cardiomyocyte cultures, which is necessary to engineer a more mature cardiomyocyte phenotype in vitro. Spectral ultrasound (SUS) may provide a way to non-invasively, non-disruptively and inexpensively monitor growth and change in cell cultures over long periods. Compared with other measurement methods, SUS as an acoustic measurement tool will not be affected by an acoustic therapy, unlike electrical measurement methods, in which motion caused by acoustic therapy can affect measurements. Further SUS has the potential to provide functional as well as morphological information in cell cultures. Human induced pluripotent stem cell cardiomyocytes (iPS-CMs) were imaged with calcium fluorescence microscopy while TUS was being applied. TUS was applied at 600 kHz and 1, 3.4 and 6 W/cm2 for a continuous 1 s pulse. Measures of the instantaneous beat frequency, repolarization rate and calcium spike amplitude were calculated from the fluorescence data. At 600 kHz, TUS at 1 and 6 W/cm2 had significant effects on the shortening of both the repolarization rate and instantaneous beat rate of the iPS-CMs (p < 0.05), while TUS at 3.4 and 6 W/cm2 had significant effects on the shortening of the calcium spike amplitude (p < 0.05). Three SUS measures and one gray-level measure were captured from the iPS-CM monolayers while they were simultaneously being imaged with calcium-labeled confocal microscopy. The gray-level measure performed the best of all SUS measures; however, it was not reliable enough to produce a consistent determination of the beat rate of the cell. Finally, SUS measures were captured using three different transducers while simultaneously applying TUS. A center-of-mass (COM) measure calculated from the wavelet transform scalogram of the time-averaged radiofrequency data revealed that SUS was able to detect a change in the frequency content of the reflected ultrasound at 1 and 6 W/cm2 before and after ultrasound application (p < 0.05), showing promise for the ability of SUS to measure changes in the beating behavior of iPS-CMs. Overall, SUS is promising as a method for constant monitoring of dynamic cell and tissue culture and growth.

Ultrasound-Enhanced Transcorneal Drug Delivery for Treatment of Fungal Keratitis.

PURPOSE: Transcorneal drug delivery is hindered by ocular physical and biochemical properties, such as tear production, the epithelial layer of the cornea, and blinking. The aim of this study was to determine whether ultrasound can be applied to increase the transcorneal drug delivery of natamycin used in the treatment of fungal keratitis without dangerously overheating the surrounding ocular tissues. METHODS: To verify the safety of various sets of ultrasound parameters, modeling studies were conducted using OnScale, an ultrasonic wave modeling software. Ultrasound parameters determined optimal for ocular tissue safety were used in a laboratory setting in a jacketed Franz diffusion cell setup. Histological images of the cross-section of the corneas used in experiments were examined for cell damage under a microscope. RESULTS: Increases in transcorneal drug delivery were seen in every treatment parameter combination when compared with the sham treatment. The highest increase was 4.0 times for 5 minutes of pulsed ultrasound at a 25% duty cycle and a frequency of 400 kHz and an intensity of 0.5 W/cm 2 with statistical significance ( P < 0.001). Histological analysis revealed structural damage only in the corneal epithelium, with most damage being at the epithelial surface. CONCLUSIONS: This study suggests that ultrasound is a safe, effective, and minimally invasive treatment method for enhancing the transcorneal drug delivery of natamycin. Further research is needed into the long-term effects of ultrasound parameters used in this study on human ocular tissues.

Feasibility of Therapeutic Ultrasound Application in Topical Scleral Delivery of Avastin.

Purpose: Macromolecules have been shown to be effective in vision-saving treatments for various ocular diseases, such as age-related macular degeneration and diabetic retinopathy. The current delivery of macromolecules requires frequent intraocular injections and carries a risk of serious adverse effects. Methods: We tested the application of therapeutic ultrasound as a minimally invasive approach for the delivery of Avastin into the diseased regions of the eye. Avastin (bevacizumab) is an anti-vascular endothelial growth factor (VEGF) antibody with a molecular weight of 149 kDa. We tested the effectiveness and safety of Avastin delivery through rabbit sclera in vitro using a standard diffusion cell model. Ultrasound at frequencies of 400 kHz or 3 MHz with an intensity of 1 W/cm2 was applied for the first 5 minutes of 1-hour drug exposure. Sham treatments mimicked the ultrasound treatments, but ultrasound was not turned on. Absorbance measurements of the receiver compartment solution were performed at 280 nm using a spectrophotometer. Results: Absorbance measurements indicated no statistical difference between the sham (n = 13) and 400 kHz ultrasound group (n = 15) in the delivery of Avastin through the sclera. However, the absorbance values were statistically different (P < 0.01) between the 3 MHz ultrasound group (0.004, n = 8) and the matched sham group (0.002, n = 7). There was 2.3 times increase in drug delivery in the 3 MHz ultrasound when compared to the corresponding sham group. Histological studies indicated no significant damage in the ultrasound-treated sclera due to ultrasound application. Conclusions: Our preliminary results provided support that therapeutic ultrasound may be effective in the delivery of Avastin through the sclera. Translational Relevance: Our study offers clinical potential for a minimally invasive retinopathy treatment.

Effect of Therapeutic Ultrasound on the Release of Insulin, Glucagon, and Alpha-Amylase from Ex Vivo Pancreatic Models.

OBJECTIVES: Our previously published studies showed the potential of therapeutic ultrasound (US) as a novel non-pharmacological alternative for the treatment of secretory deficiencies in type 2 diabetes. Despite showing enhanced insulin release from beta cells, these studies did not explore the potential effects of US treatment on other cells in the islets of Langerhans such as glucagon-secreting alpha cells or acinar cells of the exocrine pancreas. METHODS: We applied US parameters found capable of safely stimulating insulin secretion from pancreatic beta cells (f = 800 kHz, ISPTA  = 0.5-1 W/cm2 , 5 minutes) to a diced rabbit pancreas model in culture plates (n = 6 per group). Released quantities of insulin and glucagon in response to US treatment were measured by collecting aliquots of the extracellular medium prior to the start of the treatment (t = 0 minute), immediately after treatment (t = 5 minutes) and 30 minutes after the end of treatment (t = 35 minutes). Potential release of digestive enzyme alpha-amylase as a result of US treatment was evaluated in rabbit pancreas experiments. Preliminary studies were also performed in a small number of human pancreatic islets in culture plates (n = 3 per group). The general integrity of the US-treated rabbit pancreatic tissue and human pancreatic islets was evaluated through histological analysis. RESULTS: While sham-treated rabbit pancreas samples showed decreased extracellular insulin content, there was an increase in insulin release at t = 5 minutes from samples treated with US at 800 kHz and 1 W/cm2 (P <.005). Furthermore, no further insulin release was detected at t = 35 minutes. No statistically significant difference in extracellular glucagon and alpha-amylase concentrations was observed between US-treated and sham rabbit pancreas groups. Preliminary studies in human islets appeared to follow trends observed in rabbit pancreas studies. Islet and other pancreatic tissue integrity did not appear to be affected by the US treatment. CONCLUSION: A potential US-based strategy for enhanced insulin release would require optimization of insulin secretion from pancreatic beta cells while minimizing glucagon and pancreatic enzyme secretions.

Therapeutic Ultrasound-Enhanced Transcorneal PHMB Delivery In Vitro.

OBJECTIVE: Delivery of therapeutic agents to the cornea is a difficult task in the treatment of parasitic keratitis. In this study, we looked at using different combinations of ultrasound parameters to enhance corneal permeability to polyhexamethylene biguanide (PHMB), a clinically available ophthalmic antiparasitic formulation. METHODS: Permeability of PHMB was investigated in vitro using a standard diffusion cell setup. Continuous or 25% duty-cycle ultrasound was used at frequencies of 400 or 600 kHz, intensities of 0.5 or 0.8 W/cm2 , and exposure times ranging from 1 to 5 minutes. Structural changes in the cornea were examined using light microscopy. RESULTS: Ultrasound exposure produced increases in transcorneal delivery in every treatment parameter combination when compared to the sham treatment. The highest increase was 2.36 times for 5 minutes of continuous ultrasound at a frequency of 600 kHz and an intensity of 0.5 W/cm2 with statistical significance (p <.001). Histological analysis showed that ultrasound application only caused structural changes in the corneal epithelium, with most damage being at the surface layers. CONCLUSIONS: This study suggests the possibility of therapeutic ultrasound as a novel drug delivery technique for the treatment of parasitic keratitis. Further studies are needed to examine the thermal effects of these proposed ultrasound applications and the long-term viability of this treatment.

Ex Vivo Imaging of Ultrasound-Stimulated Metabolic Activity in Rat Pancreatic Slices.

Ultrasound has previously been reported to produce a reversible stimulatory effect in cultured rat beta cells. Here, we quantified and assessed dynamic 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 and 633 nm to image lipodamine 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 at 1 W/cm2 was applied in a pulsing scheme. No ultrasound (control) and glucose application experiments were performed. A difference in fluorescence signal before and after treatment application was the metric for analysis. Comparison of experimental groups using far red fluorescence revealed significant differences between all experimental groups and control in the islet (p < 0.05) and between all ultrasound experimental groups and control (p < 0.05) in pancreatic exocrine tissue. However, this difference in response between control and glucose did not exist in the exocrine tissue. We also observed using Lip-DH autofluorescence that glucose produces a significantly increased metabolic response in islet tissue compared with exocrine tissue (p < 0.05). Pulsed ultrasound appeared to increase metabolic activity in the pancreatic slice in a more consistent manner compared with continuous ultrasound application. Our results indicate that therapeutic ultrasound may have a stimulatory metabolic effect on the pancreatic islets similar to that of glucose.

Therapeutic Systems and Technologies: State-of-the-Art Applications, Opportunities, and Challenges.

In this review, we present current state-of-the-art developments and challenges in the areas of thermal therapy, ultrasound tomography, image-guided therapies, ocular drug delivery, and robotic devices in neurorehabilitation. Additionally, intellectual property and regulatory aspects pertaining to therapeutic systems and technologies are addressed.

Therapeutic Ultrasound-Induced Insulin Release in Vivo.

The tolerability and efficacy of low-frequency, low-intensity therapeutic ultrasound-induced insulin release was investigated in a pre-clinical in vivo murine model. The treatment groups received a single 5-min continuous sonication at 1 MHz and 1.0 W/cm2. Insulin and glucagon levels in the serum were determined using enzyme-linked immunosorbent assay. The pancreas was excised and sectioned for histologic analysis. In terminal studies, we observed a moderate (∼50 pM) but significant increase in blood insulin concentration in vivo immediately after sonication compared with a decrease of approximately 60 pM in sham animals (n < 6, p < 0.005). No difference was observed in the change in glucose or glucagon concentrations between groups. Comparisons of hematoxylin and eosin-stained terminal and survival pancreatic tissue revealed no visible differences or evidence of damage. This study is the first step in assessing the translational potential of therapeutic ultrasound as a treatment for early stages of type 2 diabetes.

Optimization of Ultrasound-Mediated Drug Delivery for Treatment of Onychomycosis.

BACKGROUND: Onychomycosis is a fungal nail disorder that does not have a successful cure due to the poor permeability of topical anti-fungal drugs through the nail. This study utilizes ultrasound to increase the permeability of the nail to the topical drugs currently used in clinic. The first aim of this study was to optimize ultrasonic parameters within the temperature increase limits set by the American Institute of Ultrasound in Medicine (AIUM) and the British Medical Ultrasound Society (BMUS). The second aim of the study was to evaluate the optimized parameters for a cause of action of either cavitation (the creation of micrometer pores in the nail barrier) or acoustic streaming (a steady fluid motion which may help push the drug through the nail). METHODS: Porcine and human nails are used in the five studies. PZFlex Modeling Software is used to model the temperature increase in the toe as a result of ultrasonic application and these results were used to develop the three parameters tested throughout the rest of the studies. The three parameters tested were 1 min of continuous ultrasonic application, 3 min of 50% ultrasonic application and 5 min of 50% ultrasonic application. In order to address the second aim of our research work, these three parameters were tested for the presence of streaming and cavitation. RESULTS: At the three tested parameters, the most permeation of the nail occurs with 1 min of continuous application of ultrasound to the nail. It was also found that there was limited cavitation and significant streaming at all three parameters. This suggests that streaming may be the main mechanism-of-action in ultrasound-mediated drug delivery through the nail. CONCLUSION: The parameter of 1 min of continuous ultrasonic testing will continue to be employed as the testing is moved to a rabbit model of onychomycosis.

Ultrasound-Enhanced Drug Delivery for Treatment of Acanthamoeba Keratitis.

Reaching sufficient amounts of therapeutic agents in ocular tissues is a major challenge in ophthalmology. In this study, we examined the effects of ultrasound application for delivery of polyhexamethylene biguanide for treatment of Acanthamoeba keratitis. Ultrasound intensities of 0.5 - 0.8 W/cm2 and frequencies of 400 - 600 kHz were tested with exposure durations of 1 - 5 minutes. Light microscopy was used to determine the ultrasound-induced structural changes in the cornea. All groups showed increases in drug concentration, up to 2.36 times, passing through the cornea, with the 600 kHz treatment groups reaching statistical significance. Structural changes were observed in the epithelial layer of the cornea, but the stroma and endothelium remained mostly unaffected.

Ultrasound-Induced Insulin Release as a Potential Novel Treatmentfor Type 2 Diabetes Mellitus.

Therapeutic ultrasound presents a potential novel treatment for type 2 diabetes mellitus that utilizes the non-invasive application of ultrasound energy to treat secretory defects in the earlier stages of the disease. Our previous studies have shown that ultrasound is capable of stimulating insulin release from pancreatic beta cells, safely and effectively. This study aims to both examine the calcium-dependent mechanisms of ultrasound-mediated insulin release from pancreatic beta cells using three complementary modalities - carbon fiber amperometry, ELISA studies, and Ca2+ fluorescence imaging - and to study the translational potential of therapeutic ultrasound using transgenic hyperglycemic mice for safety and efficacy studies.

Ultrasound-Enhanced Drug Delivery for Treatment of Onychomycosis.

OBJECTIVES: The aim of our study was to determine the effectiveness of using ultrasound (US) to increase the permeability of the nail, with the goal of improving outcomes in the treatment of onychomycosis. METHODS: Porcine nails were used because of their similarity to human nails. A hydrophilic blue dye was used as a drug-mimicking compound. Two sets of experiments were performed: luminosity experiments to assess the dye levels inside the nail after US and sham treatments and diffusion cell experiments for determination of changes in nail permeability due to US application. In both sets of experiments, planar US transducers were used to sonicate the nails at frequencies of 400, 600, and 800 kHz and 1 MHz, an intensity of 1 W/cm2 , and a duration of 5 min in a continuous mode. Modeling studies were also performed to assess the safety of US application to the human toe for later clinical studies. RESULTS: In the luminosity experiments, application of US at frequencies of 600 and 800 kHz led to statistically significant results (P < .05), with an increase in dye delivery into the nail of up to 95% compared to control values. The diffusion cell results found statistical significance (P < .05) at all applied frequencies, with up to a 70% increase in the nail permeability compared to the control. Safety modeling studies found a maximal temperature increase of 4.4 °C in the bone. CONCLUSIONS: Our proposed US method may offer an alternative for improved treatment of onychomycosis. The current maximal temperature increase was found to be at the safety limit, and so pulsing and other alternatives will be investigated to minimize this temperature increase.

Calcium-dependent ultrasound stimulation of secretory events from pancreatic beta cells.

BACKGROUND: Our previous studies have indicated that ultrasound can stimulate the release of insulin from pancreatic beta cells, providing a potential novel treatment for type 2 diabetes. The purpose of this study was to explore the temporal dynamics and Ca2+-dependency of ultrasound-stimulated secretory events from dopamine-loaded pancreatic beta cells in an in vitro setup. METHODS: Carbon fiber amperometry was used to detect secretion from INS-1832/13 beta cells in real time. The levels of released insulin were also measured in response to ultrasound treatment using insulin-specific ELISA kit. Beta cells were exposed to continuous wave 800 kHz ultrasound at intensities of 0.1 W/cm2, 0.5 W/cm2 and 1 W/cm2 for several seconds. Cell viability tests were done with trypan blue dye exclusion test and MTT analysis. RESULTS: Carbon fiber amperometry experiments showed that application of 800 kHz ultrasound at intensities of 0.5 and 1 W/cm2 was capable of stimulating secretory events for durations lasting as long as the duration of the stimulus. Furthermore, the amplitude of the detected peaks was reduced by 64% (p < 0.01) when extracellular Ca2+ was chelated with 10 mM EGTA in cells exposed to ultrasound intensity of 0.5 W/cm2. Measurements of released insulin in response to ultrasound stimulation showed complete inhibition of insulin secretion by chelating extracellular Ca2+ with 10 mM EGTA (p < 0.01). Viability studies showed that 800 kHz, 0.5 W/cm2 ultrasound did not cause any significant effects on viability and metabolic activity in cells exposed to ultrasound as compared to sham-treated cells. CONCLUSIONS: Our results demonstrated that application of ultrasound was capable of stimulating the release of insulin from pancreatic beta cells in a safe, controlled and Ca2+-dependent manner.

Model for Porosity Changes Occurring during Ultrasound-Enhanced Transcorneal Drug Delivery.

Ultrasound-enhanced drug delivery through the cornea has considerable therapeutic potential. However, our understanding of how ultrasound enhances drug transport is poor, as is our ability to predict the increased level of transport for given ultrasound parameters. Described here is a computational model for quantifying changes in corneal porosity during ultrasound exposure. The model is calibrated through experiments involving sodium fluorescein transport through rabbit cornea. Validation was performed using nylon filters, for which the properties are known. It was found that exposure to 800-kHz ultrasound at an intensity 2 W/cm2 for 5 min increased the porosity of the epithelium by a factor of 5. The model can be useful for determining the extent to which ultrasound enhances the amount of drug transported through biological barriers, and the time at which a therapeutic dose is achieved at a given location, for different drugs and exposure strategies.

Ultrasound Stimulation of Insulin Release from Pancreatic Beta Cells as a Potential Novel Treatment for Type 2 Diabetes.

Type 2 diabetes mellitus is a complex metabolic disease that has reached epidemic proportions in the United States and around the world. This disease is characterized by loss of insulin secretion and, eventually, destruction of insulin-producing pancreatic beta cells. Controlling type 2 diabetes is often difficult as pharmacological management routinely requires complex therapy with multiple medications, and loses its effectiveness over time. The objective of this study was to explore the effectiveness of a novel, non-pharmacological approach that uses the application of ultrasound energy to augment insulin release from rat INS 832/13 beta cells. The cells were exposed to unfocused ultrasound for 5 min at a peak intensity of 1 W/cm2 and frequencies of 400 kHz, 600 kHz, 800 kHz and 1 MHz. Insulin release was measured with enzyme-linked immunosorbent assay and cell viability was assessed via the trypan blue dye exclusion test. A marked release (approximately 150 ng/106 cells, p < 0.05) of insulin was observed when beta cells were exposed to ultrasound at 400 and 600 kHz as compared with their initial control values; however, this release was accompanied by a substantial loss in cell viability. Ultrasound application at frequencies of 800 kHz resulted in 24 ng/106 cells released insulin (p < 0.05) as compared with its unstimulated base level, while retaining cell viability. Insulin release from beta cells caused by application of 800-kHz ultrasound was comparable to that reported by the secretagogue glucose, thus operating within physiological secretory capacity of these cells. Ultrasound has potential as a novel and alternative method to current approaches aimed at correcting secretory deficiencies in patients with type 2 diabetes.

Therapeutic Modulation of Calcium Dynamics using Ultrasound and Other Energy-based Techniques.

Ultrasound, along with other types of energy-based methods, has been widely investigated for use in various therapeutic applications because of its ability to stimulate specific biological processes. Many of these processes are mediated by calcium (Ca2+) signaling, thus making modulation of Ca2+ dynamics an evident therapeutic target for energy-based techniques. Various diseases have been associated with abnormal Ca2+ signaling and could therefore benefit from therapeutic approaches trying to regulate the transport of Ca2+ across cell membranes. Here, we review published literature on the use of mechanical, electrical, magnetic and electromagnetic energy in modulating Ca2+ transients with particular emphasis on therapeutic ultrasound. We further provide brief discussions on the role of Ca2+ in living cells and the use of different experimental techniques to determine and measure its contribution to different biological processes. Finally, we explore the benefits, limitations and potential clinical applications of different energy-based modalities that can be utilized in modulation of Ca2+ signaling.