Vesna Zderic

Hemorrhage control using high intensity focused ultrasound.

Hemorrhage control is a high priority task in advanced trauma care, because hemorrhagic shock can result in less than a minute in cases of severe injuries. Hemorrhage was found to be solely responsible for 40–50% of traumatic civilian and battlefield deaths in recent years. The majority of these deaths were due to abdominal and pelvic injuries with hidden and inaccessible bleeding of solid organs such as liver, spleen, and kidneys, as well as major blood vessels. High intensity focused ultrasound (HIFU) offers a promising method for hemorrhage control. An important advantage of HIFU is that it can deliver energy to deep regions of tissue where hemorrhage is occurring, allowing cauterization at depth of parenchymal tissues, or in difficult-to-access anatomical regions, while causing no or minimal biological effects in the intervening and surrounding tissues. Moreover, HIFU can cause both thermal and mechanical effects that are shown to work synergistically for rapid hemorrhage control. The major challenges of this method are in development of bleeding detection techniques for accurate localization of the injury sites, delivery of large HIFU doses for profuse bleeding cases, and ensuring safety when critical structures are in the vicinity of the injury. Future developments of acoustic hemostasis technology are anticipated to be for applications in peripheral vascular injuries where an acoustic window is usually available, and for applications in the operating room on exposed organs.

Ocular drug delivery using 20-kHz ultrasound

The cornea is a major pathway for drug delivery to diseased eye structures. We have investigated the application of 1-s bursts of 20-kHz ultrasound, at I(SAPA) of 14 W/cm(2) (I(SATA) of 2 W/cm(2)), for enhancement of corneal permeability to glaucoma drugs of different lipophilicity (atenolol, carteolol, timolol and betaxolol). The permeability of rabbit cornea increased by 2.6 times for atenolol, 2.8 for carteolol, 1.9 for timolol and 4.4 times for betaxolol (all p-values < 0.05), after 60 min of ultrasound (US) exposure in vitro. The differences between the treatment and control experiments were statistically significant after 10 to 30 min of US exposure for all four drugs. US application appeared to produce epithelial disorganization and structural changes in the corneal stroma. Further studies are needed to determine the optimal US parameters for a safe and effective treatment.

Drug delivery into the eye with the use of ultrasound

Objective. To evaluate ultrasound enhancement of drug delivery through the cornea and the histologic appearance of the cornea up to 24 hours after treatment. Methods. Corneas were exposed to ultrasound at a frequency of 880 kHz and intensities of 0.19 to 0.56 W/cm2 (continuous mode) with an exposure duration of 5 minutes. The aqueous humor concentration of a topically applied hydrophilic dye, sodium fluorescein, was determined quantitatively in ultrasound‐ and sham‐treated rabbit eyes in vivo. Gross and light microscopic examinations were used to observe structural changes in the cornea 0 to 24 hours after ultrasound exposure. Cavitation activity was measured with a passive cavitation detector. Results. Most cells with an appearance different from that of the normal cells were present in the surface layer of the corneal epithelium. No structural changes were observed in the stroma. The increase in dye concentration in the aqueous humor (relative to sham treatment), after the simultaneous application of ultrasound and the dye solution, was 2.4 times at 0.19 W/cm2, 3.8 times at 0.34 W/cm2, and 10.6 times at 0.56 W/cm2 (P < .05). Dye delivery was found to increase with increasing ultrasound intensity, which corresponded to an increase in cavitation activity. Corneal pits, observed in the ultrasound‐treated epithelium, completely disappeared within 90 minutes. Conclusions. Application of 880‐kHz ultrasound provided up to 10‐fold enhancement in the delivery of a hydrophilic compound through the cornea while producing minor changes in the corneal epithelium.

Ultrasound-enhanced transcorneal drug delivery.

Purpose: Ultrasound has been shown to enhance, by up to 10 times, the corneal permeability to different compounds such as β-blockers and fluorescein. Here, we report on our investigation of the mechanisms of ultrasound-enhanced drug delivery through the cornea using light and electron microscopy. Methods: Enhancement of permeability for a hydrophilic compound, sodium fluorescein, in rabbit cornea in vitro was achieved using ultrasound at a frequency of 880 kHz and intensities of 0.19–0.56 W/cm2 with an exposure duration of 5 minutes. Light and electron microscopy (transmission and scanning) were used to observe ultrasound-induced structural changes in the cornea. Results: The permeability increased by 2.1, 2.5, and 4.2 times when ultrasound was applied at 0.19, 0.34, and 0.56 W/cm2, respectively (P < 0.05). The surface cells of corneal epithelium exposed to ultrasound appeared swollen and lighter in color (indications of membrane rupture) as compared with the control cells. Some of the surface epithelial cells were absent. The cells in the inner layers of the epithelium were occasionally lighter in color. Also, holes 3–10 μm in diameter were observed on the epithelial surface. No structural changes were observed in the stroma. Conclusion: Ultrasound enhancement of drug delivery through the cornea appears to result from minor structural alterations in the epithelium. Careful investigation of the recovery of cornea structure and barrier function after the ultrasound application, in vivo, is needed.

Pulsed high intensity focused ultrasound increases penetration and therapeutic efficacy of monoclonal antibodies in murine xenograft tumors

The success of radioimmunotherapy for solid tumors remains elusive due to poor biodistribution and insufficient tumor accumulation, in part, due to the unique tumor microenvironment resulting in heterogeneous tumor antibody distribution. Pulsed high intensity focused ultrasound (pulsed-HIFU) has previously been shown to increase the accumulation of (111)In labeled B3 antibody (recognizes Lewis(y) antigen). The objective of this study was to investigate the tumor penetration and therapeutic efficacy of pulsed-HIFU exposures combined with (90)Y labeled B3 mAb in an A431 solid tumor model. The ability of pulsed-HIFU (1 M Hz, spatial averaged temporal peak intensity=2685 W cm(-2); pulse repetition frequency=1 Hz; duty cycle=5%) to improve the tumor penetration and therapeutic efficacy of (90)Y labeled B3 mAb ((90)Y-B3) was evaluated in Le(y)-positive A431 tumors. Antibody penetration from the tumor surface and blood vessel surface was evaluated with fluorescently labeled B3, epi-fluorescent microscopy, and custom image analysis. Tumor size was monitored to determine treatment efficacy, indicated by survival, following various treatments with pulsed-HIFU and/or (90)Y-B3. The pulsed-HIFU exposures did not affect the vascular parameters including microvascular density, vascular size, and vascular architecture; although 1.6-fold more antibody was delivered to the solid tumors when combined with pulsed-HIFU. The distribution and penetration of the antibodies were significantly improved (p-value<0.05) when combined with pulsed-HIFU, only in the tumor periphery. Pretreatment with pulsed-HIFU significantly improved (p-value<0.05) survival over control treatments.

Annular phased-array high-intensity focused ultrasound device for image-guided therapy of uterine fibroids

An ultrasound (US), image-guided high-intensity focused ultrasound (HIFU) device was developed for noninvasive ablation of uterine fibroids. The HIFU device was an annular phased array, with a focal depth range of 30-60 mm, a natural focus of 50 mm, and a resonant frequency of 3 MHz. The in-house control software was developed to operate the HIFU electronics drive system for inducing tissue coagulation at different distances from the array. A novel imaging algorithm was developed to minimize the HIFU-induced noise in the US images. The device was able to produce lesions in bovine serum albumin-embedded polyacrylamide gels and excised pig liver. The lesions could be seen on the US images as hyperechoic regions. Depths ranging from 30 to 60 mm were sonicated at acoustic intensities of 4100 and 6100 W/cm/sup 2/ for 15 s each, with the latter producing average lesion volumes at least 63% larger than the former. Tissue sonication patterns that began distal to the transducer produced longer lesions than those that began proximally. The variation in lesion dimensions indicates the possible development of HIFU protocols that increase HIFU throughput and shorten tumor treatment times.

Optimization of pulsed focused ultrasound exposures for hyperthermia applications

Hyperthermic temperatures, with potential applications in drug/gene delivery and chemo/radio sensitization, may be generated in biological tissues by applying focused ultrasound (FUS) in pulsed mode. Here, a strategy for optimizing FUS exposures for hyperthermia applications is proposed based on theoretical simulations and in vitro experiments. Initial simulations were carried out for tissue-mimicking phantoms, and subsequent thermocouple measurements allowed for validation of the simulation results. Advanced simulations were then conducted for an ectopic, murine xenograft tumor model. The ultrasound exposure parameters investigated in this study included acoustic power (3-5 W), duty cycle (DC) (10%-50%), and pulse repetition frequency (PRF) (1-5 Hz), as well as effects of tissue perfusion. The thermocouple measurements agreed well with simulation outcomes, where differences between the two never exceeded 1.9%. Based on a desired temperature range of 39-44 °C, optimal tumor coverage (40.8% of the total tumor volume) by a single FUS exposure at 1 MHz was achieved with 4 W acoustic power, 50% DC, and 5 Hz PRF. Results of this study demonstrate the utility of a proposed strategy for optimizing pulsed-FUS induced hyperthermia. These strategies can help reduce the requirement for empirical animal experimentation, and facilitate the translation of pulsed-FUS applications to the clinic.

Prevention of post-focal thermal damage by formation of bubbles at the focus during high intensity focused ultrasound therapy

Safety concerns exist for potential thermal damage at tissue-air or tissue-bone interfaces located in the post-focal region during high intensity focused ultrasound (HIFU) treatments. We tested the feasibility of reducing thermal energy deposited at the post-focal tissue-air interfaces by producing bubbles (due to acoustic cavitation and/or boiling) at the HIFU focus. HIFU (in-situ intensities of 460-3500 W/cm2, frequencies of 3.2-5.5 MHz) was applied for 30 s to produce lesions (in turkey breast in-vitro (n = 37), and rabbit liver (n = 4) and thigh muscle in-vivo (n = 11)). Tissue temperature was measured at the tissue-air interface using a thermal (infrared) camera. Ultrasound imaging was used to detect bubbles at the HIFU focus, appearing as a hyperechoic region. In-vitro results showed that when no bubbles were present at the focus (at lower intensities of 460-850 W/cm2), the temperature at the interface increased continuously, up to 7.3 +/- 4.0 degrees C above the baseline by the end of treatment. When bubbles formed immediately after the start of HIFU treatment (at the high intensity of 3360 W/cm2), the temperature increased briefly for 3.5 s to 7.4 +/- 3.6 degrees C above the baseline temperature and then decreased to 4.0 +/- 1.4 degrees C above the baseline by the end of treatment. Similar results were obtained in in-vivo experiments with the temperature increases (above the baseline temperature) at the muscle-air and liver-air interfaces at the end of the high intensity treatment lower by 7.1 degrees C and 6.0 degrees C, respectively, as compared to the low intensity treatment. Thermal effects of HIFU at post-focal tissue-air interfaces, such as in bowels, could result in clinically significant increases in temperature. Bubble formation at the HIFU focus may provide a method for shielding the post-focal region from potential thermal damage.

Mechanical Bioeffects of Pulsed High Intensity Focused Ultrasound on a Simple Neural Model

PURPOSE To study how pressure pulses affect nerves through mechanisms that are neither thermal nor cavitational, and investigate how the effects are related to cumulative radiation-force impulse (CRFI). Applications include traumatic brain injury and acoustic neuromodulation. METHODS A simple neural model consisting of the giant axon of a live earthworm was exposed to trains of pressure pulses produced by an 825 kHz focused ultrasound transducer. The peak negative pressure of the pulses and duty cycle of the pulse train were controlled so that neither cavitation nor significant temperature rise occurred. The amplitude and conduction velocity of action-potentials triggered in the worm were measured as the magnitude of the pulses and number of pulses in the pulse trains were varied. RESULTS The functionality of the axons decreased when sufficient pulse energy was applied. The level of CRFI at which the observed effects occur is consistent with the lower levels of injury observed in this study relative to blast tubes. The relevant CRFI values are also comparable to CRFI values in other studies showing measureable changes in action-potential amplitudes and velocities. Plotting the measured action-potential amplitudes and conduction velocities from different experiments with widely varying exposure regimens against the single parameter of CRFI yielded values that agreed within 21% in terms of amplitude and 5% in velocity. A predictive model based on the assumption that the temporal rate of decay of action-potential amplitude and velocity is linearly proportional the radiation force experienced by the axon predicted the experimental amplitudes and conduction velocities to within about 20% agreement. CONCLUSIONS The functionality of axons decreased due to noncavitational mechanical effects. The radiation force, possibly by inducing changes in ion-channel permeability, appears to be a possible mechanism for explaining the observed degradation. The CRFI is also a promising parameter for quantifying neural bioeffects during exposure to pressure waves, and for predicting axon functionality.PURPOSE To study how pressure pulses affect nerves through mechanisms that are neither thermal nor cavitational, and investigate how the effects are related to cumulative radiation-force impulse (CRFI). Applications include traumatic brain injury and acoustic neuromodulation. METHODS A simple neural model consisting of the giant axon of a live earthworm was exposed to trains of pressure pulses produced by an 825 kHz focused ultrasound transducer. The peak negative pressure of the pulses and duty cycle of the pulse train were controlled so that neither cavitation nor significant temperature rise occurred. The amplitude and conduction velocity of action-potentials triggered in the worm were measured as the magnitude of the pulses and number of pulses in the pulse trains were varied. RESULTS The functionality of the axons decreased when sufficient pulse energy was applied. The level of CRFI at which the observed effects occur is consistent with the lower levels of injury observed in this study relative to blast tubes. The relevant CRFI values are also comparable to CRFI values in other studies showing measureable changes in action-potential amplitudes and velocities. Plotting the measured action-potential amplitudes and conduction velocities from different experiments with widely varying exposure regimens against the single parameter of CRFI yielded values that agreed within 21% in terms of amplitude and 5% in velocity. A predictive model based on the assumption that the temporal rate of decay of action-potential amplitude and velocity is linearly proportional the radiation force experienced by the axon predicted the experimental amplitudes and conduction velocities to within about 20% agreement. CONCLUSIONS The functionality of axons decreased due to noncavitational mechanical effects. The radiation force, possibly by inducing changes in ion-channel permeability, appears to be a possible mechanism for explaining the observed degradation. The CRFI is also a promising parameter for quantifying neural bioeffects during exposure to pressure waves, and for predicting axon functionality.

Ultrasound-Mediated Nail Drug Delivery System

A novel ultrasound‐mediated drug delivery system has been developed for treatment of a nail fungal disorder (onychomycosis) by improving delivery to the nail bed using ultrasound to increase the permeability of the nail. The slip‐in device consists of ultrasound transducers and drug delivery compartments above each toenail. The device is connected to a computer, where a software interface allows users to select their preferred course of treatment. In in vitro testing, canine nails were exposed to 3 energy levels (acoustic power of 1.2 W and exposure durations of 30, 60, and 120 seconds). A stereo ‐microscope was used to determine how much of a drug‐mimicking compound was delivered through the nail layers by measuring brightness on the cross section of each nail tested at each condition, where brightness level decreases coincide with increases in permeability. Each of the 3 energy levels tested showed statistical significance when compared to the control (P < .05) with a permeability factor of 1.3 after 30 seconds of exposure, 1.3 after 60 seconds, and 1.5 after 120 seconds, where a permeability factor of 1 shows no increase in permeability. Current treatments for onychomycosis include systemic, topical, and surgical. Even when used all together, these treatments typically take a long time to result in nail healing, thus making this ultrasound‐mediated device a promising alternative.