Transcranial focused ultrasound, pulsed at 40 Hz, activates microglia acutely and reduces Aß load chronically, as demonstrated in vivo.

OBJECTIVE: Iaccarino et al. (2016) [1] exposed 1 h of light flickering at 40 Hz to awake 5XFAD Alzheimer's Disease (AD) mouse models, generating action potentials at 40 Hz, activating ∼54% of microglia to colocalize with Aß plaque, acutely, and clearing âˆ¼ 50% of Aß plaque after seven days, but only in the visual cortex. HYPOTHESIS: Transcranially delivered, focused ultrasound (tFUS) can replicate the results of Iaccarino et al. (2016) [1] but throughout its area of application. METHODS: We exposed sedated 5XFAD mice to tFUS (2.0 MHz carrier frequency, 40 Hz pulse repetition frequency, 400 µs-long pulses, spatial peak pulse average value of 190 W/cm2). Acute studies targeted tFUS into one hemisphere of brain centered on its hippocampus for 1 h. Chronic studies targeted comparable brain in each hemisphere for 1 h/day for five days. RESULTS: Acute application of tFUS activated more microglia that colocalized with Aß plaque relative to sham ultrasound (36.0 ± 4.6% versus 14.2 ± 2.6% [mean ± standard error], z = 2.45, p < 0.014) and relative to the contralateral hemisphere of treated brain (36.0 ± 4.6% versus 14.3 ± 4.0%, z = 2.61, p < 0.009). Chronic application over five days reduced their Aß plaque burden by nearly half relative to paired sham animals (47.4 ± 5.8%, z = - 2.79, p < 0.005). CONCLUSION: Our results compare to those of Iaccarino et al. (2016) [1] but throughout the area of ultrasound-exposed brain. Our results also compare to those achieved by medications that target Aß, but over a substantially shorter period of time. The proximity of our ultrasound protocol to those shown safe for non-human primates and humans may motivate its rapid translation to human studies.

A Pre-clinical Study of the Response Threshold of Intact and Transected Nerves to Stimulation by Transcutaneous Intense Focused Ultrasound.

We used diagnostic ultrasound imaging to guide individual bursts (0.1 s) of 2 MHz intense focused ultrasound (iFU) to determine the sensitivity of intact and transected nerves. We found that all nerves had greater sensitivity to iFU stimulation than surrounding muscle. Intact nerves from healthy volunteers had less sensitivity to iFU stimulation (272 ± 35 W/cm2 [median ± standard error]) than transected nerves (19 ± 37 W/cm2). Intact, contralateral nerves of amputees dichotomized naturally into two groups-one very sensitive to iFU stimulation (6 ± 2 W/cm2) and one relatively insensitive (539 ± 19 W/cm2), compared with the intact nerves of healthy volunteers. Our study demonstrates the ability of iFU under ultrasound image guidance to stimulate deep, intact and transected peripheral nerves. It also highlights differences in the receptivity to ultrasound stimulation of the peripheral nerves of amputees versus healthy volunteers.

Transcranial and pulsed focused ultrasound that activates brain can accelerate remyelination in a mouse model of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) impacts approximately 400,000 in the United States and is the leading cause of disability among young to middle aged people in the developed world. Characteristic of this disease, myelin within generally focal volumes of brain tissue wastes away under an autoimmune assault, either inexorably or through a cycle of demyelination and remyelination. This centrally located damage produces central and peripheral symptoms tied to the portion of brain within the MS lesion site. Interestingly, Gibson and colleagues noted that optical activation of transgenically tagged central neurons increased the thickness of the myelin sheath around those neurons. Since ultrasound, delivered transcranially, can also activate brain focally, we hypothesized that ultrasound stimulation that followed the temporal pattern of Gibson et al. applied to MS lesions in a mouse model might either decelerate the demyelination phase or accelerate its remyelination phase. METHODS: We created a temporal pattern of ultrasound delivery that conformed to that of Gibson et al. and capable of activating mouse brain. We then applied ultrasound, transcranially, following that temporal pattern to separate cohorts of a mouse model of multiple sclerosis, using three different ultrasound carrier frequencies (0.625 MHz, 1.09 MHz, 2.0 MHz), during each of the demyelinating and remyelinating phases. After identifying the most promising protocol and MS brain state through qualitative analysis of myelin content, we performed additional studies for that condition then assayed for change in myelin content via quantitative analysis. RESULTS: We identified one ultrasound protocol that significantly accelerated remyelination, without damage, as demonstrated with histological analysis. CONCLUSION: MRI-guided focused ultrasound systems exist that can, in principle, deliver the ultrasound protocol we successfully tested here. In addition, MRI, as the clinical gold standard, can readily identify MS lesions. Given the relatively low intensity values of our ultrasound protocol - close to FDA limits - we anticipate that future success with this approach to MS therapy as tested using more realistic MS mouse models may one day translate to clinical trials that help address this devastating disease.

In vitro validation of endovascular Doppler-derived flow rates in models of the cerebral circulation.

This study presents validation of endovascular Doppler velocimetry-based volumetric flow rate measurements conducted in a pulsatile flow loop simulating conditions in both the internal carotid and basilar artery. In vitro models of cerebral vessels, each containing an aneurysm, were fabricated from patient anatomies extracted from 3D rotational angiography. Flow velocity measurements were collected with three different experimental techniques: an endovascular Doppler wire, Particle Image Velocimetry, and a time-resolved ultrasonic flow meter. Womersley's theory of pulsatile flow in a cylindrical vessel was used to compute time-resolved volumetric flow rates from the endovascular Doppler velocity. The volumetric flow rates computed from the Doppler measurements were compared to those from the Particle Image Velocimetry profile measurements, and the direct measurements from the ultrasonic flow meter. The study establishes confidence intervals for any systematic or random errors associated with the wire-derived flow rates as benchmarked to the other two modalities. There is an approximately 10% random error in the Doppler-derived peak and time-averaged flow rates. There is a measurable uniform bias, about 15% too low, in the time-averaged Doppler-derived flow rates. There is also a small proportional bias in the peak systolic Doppler-derived flow rates. Potential sources of error are also discussed.

Rediscovering the wound hematoma as a site of hemostasis during major arterial hemorrhage.

BACKGROUND: Treatments for major internal bleeding after injury include permissive hypotension to decrease the rate of blood loss, intravenous infusion of plasma or clotting factors to improve clot formation, and rapid surgical hemostasis or arterial embolization to control bleeding vessels. Yet, little is known regarding major internal arterial hemostasis, or how these commonly used treatments might influence hemostasis. OBJECTIVES: (i) To use a swine model of femoral artery bleeding to understand the perivascular hemostatic response to contained arterial hemorrhage. (ii) To directly confirm the association between hemodynamics and bleeding velocity. (iii) To observe the feasibility of delivering an activated clotting factor directly to internal sites of bleeding using a simplified angiographic approach. METHODS: Ultrasound was used to measure bleeding velocity and in vivo clot formation by elastography in a swine model of contained femoral artery bleeding with fluid resuscitation. A swine model of internal pelvic and axillary artery hemorrhage was also used to demonstrate the feasibility of local delivery of an activated clotting factor. RESULTS: In this model, clots formed slowly within the peri-wound hematoma, but eventually contained the bleeding. Central hemodynamics correlated positively with bleeding velocity. Infusion of recombinant human activated factor VII into the injured artery near the site of major internal hemorrhage in the pelvis and axillae was feasible. CONCLUSIONS: We rediscovered that clot formation within the peri-wound hematoma is an integral component of hemostasis and a feasible target for the treatment of major internal bleeding using activated clotting factors delivered using a simplified angiographic approach.

Cerebral aneurysms treated with flow-diverting stents: computational models with intravascular blood flow measurements.

BACKGROUND AND PURPOSE: Computational fluid dynamics modeling is useful in the study of the hemodynamic environment of cerebral aneurysms, but patient-specific measurements of boundary conditions, such as blood flow velocity and pressure, have not been previously applied to the study of flow-diverting stents. We integrated patient-specific intravascular blood flow velocity and pressure measurements into computational models of aneurysms before and after treatment with flow-diverting stents to determine stent effects on aneurysm hemodynamics. MATERIALS AND METHODS: Blood flow velocity and pressure were measured in peri-aneurysmal locations by use of an intravascular dual-sensor pressure and Doppler velocity guidewire before and after flow-diverting stent treatment of 4 unruptured cerebral aneurysms. These measurements defined inflow and outflow boundary conditions for computational models. Intra-aneurysmal flow rates, wall shear stress, and wall shear stress gradient were calculated. RESULTS: Measurements of inflow velocity and outflow pressure were successful in all 4 patients. Computational models incorporating these measurements demonstrated significant reductions in intra-aneurysmal wall shear stress and wall shear stress gradient and a trend in reduced intra-aneurysmal blood flow. CONCLUSIONS: Integration of intravascular dual-sensor guidewire measurements of blood flow velocity and blood pressure provided patient-specific computational models of cerebral aneurysms. Aneurysm treatment with flow-diverting stents reduces blood flow and hemodynamic shear stress in the aneurysm dome.

Activation, aggregation and adhesion of platelets exposed to high-intensity focused ultrasound.

Using platelet-rich plasma, we investigated the effect of 1.1-MHz continuous wave high-intensity focused ultrasound (HIFU) on platelet activation, aggregation and adhesion to a collagen-coated surface. Platelets were exposed for durations of 10-500 s at spatial average intensities of up to 4860 W/cm(2). To avoid heating effects, the average temperature in the HIFU tank was maintained at 33.8 +/- 4.0 degrees C during platelet experiments. Flow cytometry, laser aggregometry, environmental scanning electron microscopy and passive cavitation detection were used to observe and to quantify platelet activation, aggregation, adhesion to a collagen-coated surface and associated cavitation. It was determined that HIFU can activate platelets, stimulate them to aggregate and promote their adherence to a collagen-coated surface. In principle, HIFU can stimulate primary, or platelet-related, hemostasis. Cavitation was monitored by a passive cavitation detector during aggregation trials and was quantified to provide a relative measure of the amount of cavitation that occurred in each aggregation trial. Regression analysis shows a weak correlation (r(2) = 0.11) between aggregation and ultrasound intensity, but a substantial correlation (r(2) = 0.76) between aggregation and cavitation occurrence.

Self-assembled molecular structures as ultrasonically-responsive barrier membranes for pulsatile drug delivery.

Noninvasive ultrasound has been shown to increase the release rate on demand from drug delivery systems; however, such systems generally suffer from background drug leaching. To address this issue, a drug-containing polymeric monolith coated with a novel ultrasound-responsive coating was developed. A self-assembled molecular structure coating based on relatively impermeable, ordered methylene chains forms an ultrasound-activated on-off switch in controlling drug release on demand, while keeping the drug inside the polymer carrier in the absence of ultrasound. The orderly structure and molecular orientation of these C12 n-alkyl methylene chains on polymeric surfaces resemble self-assembled monolayers on gold. Their preparation and characterization have been published recently (Kwok et al. [Biomacromolecules 2000;1(1):139-148]). Ultrasound release studies showed that a copolymer of 2-hydroxyethyl methacrylate and ethylene glycol dimethacrylate (MW 400) coated with such an ultrasound-responsive membrane maintained sufficient insulin for multiple insulin delivery, compared with a substantial burst release during the first 2 h from uncoated samples. With appropriate surface coating coverage, the background leach rate can be precisely controlled. The biological activity of the insulin releasate was tested by assessing its ability to regulate [C14]-deoxyglucose uptake in 3T3-L1 adipocyte cells in a controlled cell culture environment. Uptake triggered by released insulin was comparable to that of the positive insulin control. The data demonstrate that the released insulin remains active even after the insulin had been exposed to matrix synthesis and the methylene chain coating process.

Acceleration of recovery after injury to the peripheral nervous system using ultrasound and other therapeutic modalities.

Taken together, these studies show the promise of various therapeutic modalities for the noninvasive treatment of peripheral nerve injury. Further progress on these promising methods requires determining the biologic mechanisms responsible for the ability of these modalities to enhance peripheral nerve recovery. Necessary investigations include validation or refutation of the hypothesis that these therapies act on various aspects of the natural healing process. Examples include cellular and molecular processes involved in promoting Wallerian degeneration and the rate and specificity of axonal regeneration and remyelination and muscle reinnervation, processes that are distributed between the regenerating nerve itself, the pathway of the regenerating axon, and the target of the regenerating nerve. An increased understanding of the biologic mechanisms underlying the enhancement of peripheral nerve recovery after injury would lend greater insight into the cellular and molecular mechanisms involved in successful nerve regeneration and muscle reinnervation. This increased understanding may also result in clinically beneficial treatments for peripheral nerve disorders.

Ultrasound accelerates functional recovery after peripheral nerve damage.

OBJECTIVE: Axonal injury in the peripheral nervous system is common, and often it is associated with severe long-term personal and societal costs. The objective of this study is to use an animal model to demonstrate that transcutaneous ultrasound can accelerate recovery from an axonotmetic injury. METHODS: The sciatic nerve of adult male Lewis rats was crushed in the right midthigh to cause complete distal degeneration of axons yet maintain continuity of the nerve. Beginning 3 days after surgery, various transcutaneous ultrasound treatments or sham treatments were applied 3 days per week for 30 days to the crush site of rats that were randomly assigned to two groups. In the preliminary experiments, there were three animals in each ultrasound group and two control animals. In the final experiment, there were 22 animals in the ultrasound group and 20 animals in the control group. Recovery was assessed by use of a toe spread assay to quantify a return to normal foot function in the injured leg. Equipment included a hand-held transducer that emitted continuous-wave ultrasound. The most successful ultrasound protocol had a spatial peak, time-averaged intensity of 0.25 W/cm2 operated at 2.25 MHz for 1 minute per application. RESULTS: Rats subjected to the most successful ultrasound protocol showed a statistically significant acceleration of foot function recovery starting 14 days after injury versus 18 days for the control group. Full recovery by the ultrasound group occurred before full recovery by the control group. CONCLUSION: Transcutaneous ultrasound applied to an animal model of axonotmetic injury accelerated recovery. Future studies should focus on identification of the mechanism(s) by which ultrasound creates this effect, as a prelude to optimization of the protocol, demonstration of its safety, and its eventual application to humans.

Thresholds for inertial cavitation in albunex suspensions under pulsed ultrasound conditions.

Stabilized microbubbles used as echo-contrast agents can be destroyed by ultrasonic irradiation. We have identified two pressure thresholds at which these microbubbles undergo inertial cavitation (here, defined as the collapse of gas bubbles followed by emission of an acoustic broadband noise). The first threshold (P1) corresponds to the pressure at which all the microbubbles in a cavitation field lose their property as an effective scatterer because of fragmentation or deflation. The second threshold (P2) is associated with the acoustic reactivation of the remnants of the contrast agents and is related to the onset of more violent inertial cavitation. P1 and P2 were measured as a function of the concentration of Albunex (Molecular Biosystems Inc., San Diego, CA) contrast agent, the number of transmitting acoustic cycles, and the pulse repetition frequency (PRF). The ultrasound frequency used was 1.1 MHz, and the peak negative acoustic pressures ranged from 0 to 8 MPa. Our results, measured in Isoton II (Coulter Diagnostics, Miami, FL) and whole blood solutions, showed that P1 increased with increasing Albunex concentration and decreased with increasing PRF, whereas P2 decreased with increasing Albunex concentration and was independent of the PRF. Both P1 and P2 decreased with increasing number of acoustic cycles N for N < 10 and were independent of the number of cycles for N > 10. Ultrasound images of Albunex acquired by a commercial scanner showed echo enhancement not only at pressure levels below P1 but also at levels above P2. The threshold P2 was achieved at ultrasound energies above the diagnostic level. Inertial cavitation produced at P2 was associated with a higher level of hemolysis compared with P1. The results of this investigation have potential significance for both diagnostic and therapeutic ultrasound applications.

Numerical simulations of heating patterns and tissue temperature response due to high-intensity focused ultrasound.

The results of this paper show-for an existing high intensity, focused ultrasound (HIFU) transducer-the importance of nonlinear effects on the space/time properties of wave propagation and heat generation in perfused liver models when a blood vessel also might be present. These simulations are based on the nonlinear parabolic equation for sound propagation and the bio-heat equation for temperature generation. The use of high initial pressure in HIFU transducers in combination with the physical characteristics of biological tissue induces shock formation during the propagation of a therapeutic ultrasound wave. The induced shock directly affects the rate at which heat is absorbed by tissue at the focus without significant influence on the magnitude and spatial distribution of the energy being delivered. When shocks form close to the focus, nonlinear enhancement of heating is confined in a small region around the focus and generates a higher localized thermal impact on the tissue than that predicted by linear theory. The presence of a blood vessel changes the spatial distribution of both the heating rate and temperature.

Surface modification of polymers with self-assembled molecular structures: multitechnique surface characterization.

A simple, one-step procedure for generating ordered, crystalline methylene chains on polymeric surfaces via urethane linkages was developed. The reaction of dodecyl isocyanate with surface hydroxyl functional groups, catalyzed by dibutyltin dilaurate, formed a predominantly all-trans, crystalline structure on a cross-linked poly(2-hydroxyethyl methacrylate) (pHEMA) substrate. Allophanate side-branching reactions were not observed. Both X-ray photoelectron spectrocopy and time-of-flight secondary ion mass spectrometry show that the surface reaction reached saturation after 30 min at 60 degrees C. Unpolarized Fourier transform infrared-attenuated total reflection showed that, after 30 min, the stretching frequencies, vCH2,asym and vCH2,sym, decreased and approached 2920 and 2850 cm-1, indicative of a crystalline phase. The distance between two hydroxyl groups is roughly 4 A. A tilt angle of 33.5 degrees +/- 2.4 degrees was estimated by dichoric ratios measured in polarized ATR according to the two-phase and Harrick thin film approximations. The findings reported here are significant in that the possibilities for using structures similar to self-assembled monolayers (SAMs) are expanded beyond the rigid gold and silicon surfaces used through most of the literature. Thus, SAMs, biomimetics for ordered lipid cell wall structures, can be applied to real-world biomedical polymers to modify biological interactions. The terminal groups of the SAM-like structure can be further functionalized with biomolecules or antibodies to develop surface-based diagnostics, biosensors, or biomaterials.

A review and examination of ultrasound for lipoplasty.

Ultrasonic lipoplasty occurs through a complex and describable but currently unquantified series of processes. Ample opportunity exists for the creation of deleterious effects, such as burning tissue and destroying blood vessels and nerves; however, as reported by others, the skilled user seems able to avoid these problems. The possibility of free radical-induced cancer being caused by ultrasonic lipoplasty seems extremely unlikely to the authors. Still, the authors cannot categorically rule out the possibility of significant long-term, harmful, biologic effects caused by free-radical production. All the authors have been able to do is produce an extremely generous upper bound on hydrogen peroxide production and note the uphill climb that any free radical would have to surmount to do lasting damage. Also, users should ensure that no preexisting cancers are present in the fatty tissue or adjoining tissue. Finally, these and other issues, concerns, and hypotheses would all benefit from significant tests on appropriate animal models.

Effect of high-intensity focused ultrasound on whole blood with and without microbubble contrast agent.

Using human whole blood samples with and without contrast agent (CA), we evaluated the effect of exposures to focused, continuous wave (CW) 1.1-MHz ultrasound for durations of 10 ms to 1 s at spatial average intensities of 560 to 2360 W/cm2. Cavitation was monitored with a passive cavitation detector and hemolysis was determined with spectroscopy. In whole blood alone, no significant cavitation, heating or hemolysis was detected at any exposure condition. Conversely, cavitation and hemolysis, but not heating, were detected in whole blood with CA. A CA concentration as low as 0.28 microL CA per mL whole blood at an intensity of 2360 W/cm2 for 1 s resulted in measurable cavitation and a 6-fold increase in hemolysis compared to shams. Cavitation and hemolysis increased proportional to the concentration of CA and duration of exposure. In samples containing 4.2 microL CA per mL whole blood exposed for 1 s, a threshold was seen at 1750 W/cm2 where cavitation and hemolysis increased 10-fold compared to exposures at lower intensities. HIFU exposure of whole blood containing CA leads to significant hemolysis in vitro and may lead to clinically significant hemolysis in vivo.