Computational hemodynamic simulations of cerebral aneurysms have traditionally relied on stereotypical boundary conditions (such as blood flow velocity and blood pressure) derived from published values as patient-specific measurements are unavailable or difficult to collect. However, controversy persists over the necessity of incorporating such patient-specific conditions into computational analyses. We perform simulations using both endovascularly-derived patient-specific and typical literature-derived inflow and outflow boundary conditions. Detailed three-dimensional anatomical models of the cerebral vasculature are developed from rotational angiography data, and blood flow velocity and pressure are measured in situ by a dual-sensor pressure and velocity endovascular guidewire at multiple peri-aneurysmal locations in 10 unruptured cerebral aneurysms. These measurements are used to define inflow and outflow boundary conditions for computational hemodynamic models of the aneurysms. The additional in situ measurements which are not prescribed in the simulation are then used to assess the accuracy of the simulated flow velocity and pressure drop. Simulated velocities using patient-specific boundary conditions show good agreement with the guidewire measurements at measurement locations inside the domain, with no bias in the agreement and a random scatter of ≈25%. Simulated velocities using the simplified, literature-derived values show a systematic bias and over-predicted velocity by ≈30% with a random scatter of ≈40%. Computational hemodynamics using endovascularly measured patient-specific boundary conditions have the potential to improve treatment predictions as they provide more accurate and precise results of the aneurysmal hemodynamics than those based on commonly accepted reference values for boundary conditions.

Transcranial ultrasound can alter brain function transiently and nondestructively, offering a new tool to study brain function now and inform future therapies. Previous research on neuromodulation implemented pulsed low-frequency (250–700 kHz) ultrasound with spatial peak temporal average intensities (I_SPTA) of 0.1–10 W/cm². That work used transducers that either insonified relatively large volumes of mouse brain (several mL) with relatively low-frequency ultrasound and produced bilateral motor responses, or relatively small volumes of brain (on the order of 0.06 mL) with relatively high-frequency ultrasound that produced unilateral motor responses. This study seeks to increase anatomical specificity to neuromodulation with modulated focused ultrasound (mFU). Here, "modulated" means modifying a focused 2-MHz carrier signal dynamically with a 500-kHz signal as in vibro-acoustography, thereby creating a low-frequency but small volume (approximately 0.015 mL) source of neuromodulation.

Application of transcranial mFU to lightly anesthetized mice produced various motor movements with high spatial selectivity (on the order of 1 mm) that scaled with the temporal average ultrasound intensity. Alone, mFU and focused ultrasound (FUS) each induced motor activity, including unilateral motions, though anatomical location and type of motion varied. Future work should include larger animal models to determine the relative efficacy of mFU versus FUS. Other studies should determine the biophysical processes through which they act. Also of interest is exploration of the potential research and clinical applications for targeted, transcranial neuromodulation created by modulated focused ultrasound, especially mFU's ability to produce compact sources of ultrasound at the very low frequencies (10–100s of Hertz) that are commensurate with the natural frequencies of the brain.

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.

Mild traumatic brain injury (mTBI) is considered the"signature injury" of combat veterans that have served during the wars in Iraq and Afghanistan. This prevalence of mTBI is due in part to the common exposure to high explosive blasts in combat zones. In addition to the threats of blunt impact trauma caused by flying objects and the head itself being propelled against objects, the primary blast overpressure (BOP) generated by high explosives is capable of injuring the brain. Compared to other means of causing TBI, the pathophysiology of mild-to-moderate BOP is less well understood. To study the consequences of BOP exposure in mice, we employed a well-established approach using a compressed gas-driven shock tube that recapitulates battlefield-relevant open-field BOP. We found that 24 hours post-blast a single mild BOP provoked elevation of multiple phospho- and cleaved-tau species in neurons, as well as elevating manganese superoxide-dismutase (MnSOD or SOD2) levels, a cellular response to oxidative stress. In hippocampus, aberrant tau species persisted for at least 30 days post-exposure, while SOD2 levels returned to sham control levels. These findings suggest that elevated phospho- and cleaved-tau species may be among the initiating pathologic processes induced by mild blast exposure. These findings may have important implications for efforts to prevent blast-induced insults to the brain from progressing into long-term neurodegenerative disease processes.

Previous studies have observed that individual pulses of intense focused ultrasound (iFU) applied to inflamed and normal tissue can generate sensations, where inflamed tissue responds at a lower intensity than normal tissue. It was hypothesized that successively applied iFU pulses will generate sensation in inflamed tissue at a lower intensity and dose than application of a single iFU pulse. This hypothesis was tested using an animal model of chronic inflammatory pain, created by injecting an irritant into the rat hind paw. Ultrasound pulses were applied in rapid succession or individually to rats' rear paws beginning at low peak intensities and progressing to higher peak intensities, until the rats withdrew their paws immediately after iFU application. Focused ultrasound protocols consisting of successively and rapidly applied pulses elicited inflamed paw withdrawal at lower intensity and estimated tissue displacement values than single pulse protocols. However, both successively applied pulses and single pulses produced comparable threshold acoustic dose values and estimates of temperature increases. This raises the possibility that temperature increase contributed to paw withdrawal after rapid iFU stimulation. While iFU-induction of temporal summation may also play a role, electrophysiological studies are necessary to tease out these potential contributors to iFU stimulation.

A diagnosis criterion is proposed for noninvasive grading of burn injuries using terahertz radiation. Experimental results are presented from in vivo terahertz time-domain spectroscopy of second- and third-degree wounds, which are obtained in a 72-hour animal study. During this period, the change in the spectroscopic response of the burned tissue is studied. It is shown that terahertz waves are sensitive not only to the postburn formation of interstitial edema, but also to the density of skin structures derived from image processing analysis of histological sections. Based on these preliminary results, it is suggested that the combination of these two effects, as probed by terahertz spectroscopy of the tissue, may ultimately be used to differentiate partial-thickness burns that will naturally heal from those that will require surgical intervention.

Ischemia, edema, elevated intracranial pressure, and reduced blood flow can occur in the brain as a result of ischemic stroke, including contralateral to the stroke via a process known as diaschisis. In this study, ultrasound elastography, an imaging process sensitive to the stiffness of tissue, including its relative fluid content, was used to study changes in the stiffness of individual cerebral hemispheres after transient ischemic injury. Elastographic images of mouse brains were collected 24 and 72 hours after middle cerebral artery occlusion. The shear moduli of both ipsilateral and contralateral brain hemispheres for these mice were measured and compared to corresponding values of control animals.

At 24 hours (but not 72 hours) after induction of ischemic stroke, there was a significant decrease in the shear modulus in the ipsilateral hemisphere (P < .01) and a significant increase in the shear modulus in the contralateral hemisphere compared to that of control animals (P < .01). Significant differences were also evident between ipsilateral and contralateral shear modulus values at 24 and 72 hours after infarction (P < .01 for both). The differences between intrahemispheric averages of shear moduli of the brains of animals with stroke at 24 and 72 hours after stroke induction likely reflect the initial formation of edema and reduction of cerebral blood flow known to develop ipsilateral to ischemic infarction, the known transient increase in intracranial pressure, as well as the known initial reduction of blood flow and subsequent development of edema in the contralateral hemisphere (diaschisis). Thus, elastography offers a possible method to detect subtle changes in brain after ischemic stroke.

BACKGROUND: It has been hypothesized that the critical closing pressure of cerebral circulation, or zero-flow pressure (ZFP), can estimate intracranial pressure (ICP). One ZFP estimation method used extrapolation of arterial blood pressure as against blood-flow velocity. The aim of this study was to improve ICP predictions. METHODS: Two revisions have been considered: (1) the linear model used for extrapolation is extended to a nonlinear equation; and (2) the parameters of the model are estimated by an alternative criterion (not least squares). The method is applied to data on transcranial Doppler measurements of blood-flow velocity, arterial blood pressure, and ICP from 104 patients suffering from closed traumatic brain injury, sampled across the United States and England. RESULTS: The revisions lead to qualitative (eg, precluding negative ICP) and quantitative improvements in ICP prediction. While moving from the original to the revised method, the ±2 SD of the error is reduced from 33 to 24 mm Hg, and the root-mean-squared error is reduced from 11 to 8.2 mm Hg. The distribution of root-mean-squared error is tighter as well; for the revised method the 25th and 75th percentiles are 4.1 and 13.7 mm Hg, respectively, as compared with 5.1 and 18.8 mm Hg for the original method. CONCLUSIONS: Proposed alterations to a procedure for estimating ZFP lead to more accurate and more precise estimates of ICP, thereby offering improved means of estimating it noninvasively. The quality of the estimates is inadequate for many applications, but further work is proposed, which may lead to clinically useful results.

Quantifying pain through assay of a human's or animal's response to a known stimulus as a function of time of day is a critical means of advancing chronotherapeutic pain management. Current methods for quantifying pain, even in the context of etiologies involving deep tissue, generally involve stimulation by quantifiable means of either cutaneous (heat-lamp tests, electrical stimuli) or both cutaneous and subcutaneous tissue (von Frey hairs, tourniquets, etc.) or study of proxies for pain (such as stress, via assay of cortisol levels). In this study, we evaluate the usefulness of intense focused ultrasound (iFU), already shown to generate sensations and other biological effects deep to the skin, as a means of quantifying deep diurnal pain using a standard animal model of inflammation. Beginning 5 days after injection of Complete Freund's Adjuvant into the plantar surface of the rat's right hind paw to induce inflammation, the rats were divided into two groups, the light-phase test group (09:00-18:00h) and the dark-phase test group (23:00-06:00h), both of which underwent iFU application deep to the skin. We used two classes of iFU protocol, motivated by the extant literature. One consisted of a single pulse (SP) lasting 0.375s. The other, a multiple pulse (MP) protocol, consisted of multiple iFU pulses each of length 0.075s spaced 0.075s apart. We found the night group's threshold for reliable paw withdrawal to be significantly higher than that of the day group as assayed by each iFU protocol. These results are consistent with the observation that the response to mechanical stimuli by humans and rodents display diurnal variations, as well as the ability of iFU to generate sensations via mechanical stimulation. Since iFU can provide a consistent method to quantify pain from deep, inflamed tissue, it may represent a useful adjunct to those studying diurnal pain associated with deep tissue as well as chronotherapeutics targeting that pain.

Objective.
Potential peripheral sources of pain from subcutaneous tissue can require invasive evocative tests for their localization and assessment. Here, we describe studies whose ultimate goal is development of a noninvasive evocative test for subcutaneous, painful tissue.

Design.
We used a rat model of a focal and subcutaneous neuroma to test the hypothesis that intense focused ultrasound can differentiate focal and subcutaneous neuropathic tissue from control tissue. To do so, we first applied intense focused ultrasound (2 MHz, with individual pulses of 0.1 second in duration) to the rat's neuroma while the rat was under light anesthesia. We started with low values of intensity, which we increased until intense focused ultrasound stimulation caused the rat to reliably flick its paw. We then applied that same intense focused ultrasound protocol to control tissue away from the neuroma and assayed for the rat's response to that stimulation.

Results.
Intense focused ultrasound of sufficient strength (I_SATA of 600 ± 160 W/cm²) applied to the neuroma caused the rat to flick its paw, while the same intense focused ultrasound applied millimeters to a centimeter away failed to induce a paw flick.

Conclusion.
Successful stimulation of the neuroma by intense focused ultrasound required colocalization of the neuroma and intense focused ultrasound supporting our hypothesis.

We tested the hypothesis that neuropathic tissue is more sensitive to stimulation by intense focused ultrasound (iFU) than control tissue. We created a diffusely neuropathic paw in rats via partial ligation of the sciatic nerve, whose sensitivity to iFU stimulation we compared with sham-surgery and normal control paws. We then applied increasing amounts of iFU (individual 0.2 s pulses at 1.15 MHz) to the rats' paws, assaying for their reliable withdrawal from that stimulation. Neuropathic rats preferentially withdrew their injured paw from iFU at smaller values of iFU intensity (84.2 W/cm² ± 25.5) than did sham surgery (97.7 W/cm² ± 11.9) and normal control (> 223 W/cm²) animals, with greater sensitivity and specificity (85% for neuropathic rats and 50% each of sham surgery and normal control rats). These results directly support our hypothesis as well as Gavrilov's idea that doctors may some day use iFU stimulation to diagnose patients with neuropathies.

We demonstrate the application of THz-TDS in characterizing the severity of burn injuries in a live animal model. 2nd and 3rd degree burns were studied immediately and 72 hours post-burn. A new diagnosis criterion was verified against histopathology.

Sensations generated by intense focused ultrasound (iFU) can occur cutaneously and/or at depth, in contrast to other forms of stimulation (e.g., heat, electricity), whose action usually occurs only at the skin surface, or mechanical stimulation (e.g., von Frey hairs, calibrated forceps, tourniquets) that compress and thus stimulate all tissue. Previous work on iFU stimulation has led to the hypothesis that the tactile basis of iFU stimulation should correlate with the density of mechanoreceptors at the site of iFU stimulation. Here we tested that hypothesis, correlating a 'two-point' neurological examination-a standard measure of superficial mechanoreceptor density-with the intensity of superficially applied iFU necessary to generate sensations with high sensitivity and specificity. We applied iFU at 1.1 MHz for 0.1 s to the fingertip pads of 17 test subjects in a blinded fashion and escalated intensities until they consistently observed iFU-induced sensations. Most test subjects achieved high values of sensitivity and specificity, doing so at values of spatially and temporally averaged intensity measuring <100 W/cm². Moreover, the test subjects' sensitivity to iFU stimulation correlated with the density of mechanoreceptors as determined by a standard two-point discrimination neurological examination, consistent with earlier hypotheses.

We present sub-millimeter wave reflectometry of an experimental rat skin burn model obtained by the Terahertz Time-Domain Spectroscopy (THz-TDS) technique. Full thickness burns, as confirmed by histology, were created on rats (n = 4) euthanized immediately prior to the experiments. Statistical analysis shows that the burned tissue exhibits higher reflectivity compared to normal skin over a frequency range between 0.5 and 0.7 THz (p < 0.05), likely due to post-burn formation of interstitial edema. Furthermore, we demonstrate that a double Debye dielectric relaxation model can be used to explain the terahertz response of both normal and less severely burned rat skin. Finally, our data suggest that the degree of conformation between the experimental burn measurements and the model for normal skin can potentially be used to infer the extent of burn severity.

Vibro-acoustography (VA) is an ultrasound interrogation and imaging technique with a variety of applications. Here it was used to identify optimal parameters for detecting and imaging kidney stones in phantoms. The parameters varied included the difference frequency and the position in time of the analysis window used for image construction. Experiments in a water tank were conducted using a focused PVDF membrane hydrophone (receiver) placed in a central opening of an annular, dual element transducer (source), itself mounted on a translation stage. Our source consisted of 90-ms pulses with a center frequency of 2.0 MHz and difference frequencies between 50 and 350 kHz, applied both on and off stone. Variations in the amplitude of the measured ultrasound backscatter and acoustic emissions as a function of difference frequency, between signals from stone and phantom, guided the choice of imaging parameters. The results were detailed images of renal stones measuring 10 dB above the background tissue. These findings suggest that spectral information from the scattering and reverberation of VA induced ultrasound can be used to guide the interrogation and imaging of kidney stones.

The accuracy rates of the clinical assessment techniques used in grading burn injuries remain significantly low for partial thickness burns. In this paper, we present experimental results from terahertz characterization of 2nd and 3rd degree burn wounds induced on a rat model. Reflection measurements were obtained from the surface of both burned and normal skin using pulsed terahertz spectroscopy. Signal processing techniques are described for interpretation of the acquired terahertz waveform and differentiation of burn wounds. Furthermore, the progression of burn injuries is shown by comparison between acute characterization and 72-hours survival studies. While the water content of healthy and desiccated skin has been considered as a source of terahertz signal contrast, it is demonstrated that other biological effects such as formation of post-burn interstitial edema as well as the density of the discrete scattering structures in the skin (such as hair follicles, sweat glands, etc.) play a significant role in the terahertz response of the burn wounds.

PURPOSE: To investigate in vitro the use of ultrasound in a power toothbrush to aid in the removal of dental plaque biofilm without bristle contact.

METHODS: Dental plaque was modeled using Streptococcus mutans biofilm adherent to hydroxyapatite disks. Treatment arms included positive and negative controls, disks with and without biofilm, respectively. Power toothbrush modes of action tested included a toothbrush with sonic and ultrasonic action (ULT), the same toothbrush with only sonic action (ULN), a sonic toothbrush (SON) and a rotating/oscillating toothbrush (OSC). The active element of the toothbrushes (bristles or point of ultrasound emission) was immersed in toothpaste slurry and held 3 mm away from the disk surface. Treatment included activation of the toothbrush mode of action for 5 seconds. Control disks were exposed to the same fluid environment but not exposed to a power toothbrush. After treatment, biofilm present on the disks was stained using a red dental plaque disclosing solution. Photographs were then taken and the presence of biofilm assessed using digital image analysis. For each disk a normalized pixel volume, related to the presence of biofilm corrected for lighting, was determined. Statistical testing was done with a one-way ANOVA and a Bonferroni post hoc test.

RESULTS: Normalized pixel volumes (mean /- standard deviation) were 0.428 (0.010) for the negative control and 1.022 (0.040) for the positive control. Normalized pixel volumes for the power toothbrush modes of action were 0.641 (0.075) for ULT, 0.972 (0.027) for ULN, 0.921 (0.010) for SON and 0.955 (0.025) for OSC. Statistical analysis showed a significant treatment effect (P<0.001). All power toothbrush modes of action exhibited some biofilm removal without bristle contact in this in vitro assay. Of the modes of action tested, the combined sonic and ultrasonic mode of action (ULT) removed the greatest amount of biofilm from the disk surfaces. The same toothbrush when tested with (ULT) and without (ULN) ultrasound showed a greater amount of biofilm removed when ultrasound was present.

Efficacious, daily oral health care represents an important part of maintaining the overall health of individuals. Here we sought to develop and demonstrate the usefulness of a new power toothbrush, one that incorporated both sonic processes (in the form of rapid bristle motion that directly removes plaque as well as generates bubbles) and ultrasound (sourced from the brush head, that activated the bubbles). We adapted a set of existing power toothbrushes to incorporate an ultrasound source with sufficient controls to explore the effects of different ultrasound parameters on plaque removal in vitro and in vivo. The combination of sonic and ultrasound physics removed plaque in vitro in a synergistic manner, likely through the action of cavitation. In vivo, the Ultreo toothbrush — a commercial version of this brush — removed comparable amounts of plaque to another commercial brush in half the time, and removed more plaque than manual toothbrushes. A combination of sonic bristle motion and ultrasound-facilitated cavitation appears to produce superior plaque removal compared to existing, sonic only, or manual tooth brushes.

Pressure within the cranium (intracranial pressure, or "ICP") represents a vital clinical variable whose assessment — currently via invasive means — and integration into a clinical exam constitutes a necessary step for adequate medical care for those patients with injured brains. In the present work we sought to develop a non-invasive way of predicting this variable and its corollary — cerebral perfusion pressure (CPP), which equals ICP minus arterial blood pressure (ABP).

We collected transcranial Doppler (TCD), invasive ICP and ABP data from patients at a variety of hospitals. We developed a series of regression-based statistical algorithms for subsets of those patients sorted by etiology with the goal of predicting ICP and CPP. We could discriminate between high and low values of ICP (above/below 20 mmHg) with sensitivities and specificities generally greater than 70 percent, and predict CPP within ±5 percent, for patients with traumatic brain injury. TCD and invasive ABP data can be translated into useful measures of ICP and CPP. Future work will target use of non-invasive ABP data, automation of TCD data acquisition, and improvement in algorithm performance.

Localizing painful tissue (pain generators) is central to medicine. Here we sought to demonstrate that intense focused ultrasound (iFU) can successfully differentiate normal from painful tissue. Rats were injected with complete Freunds adjuvant in a hind paw to create an inflammatory injury, or underwent a surgical procedure that damaged the nerve enervating the paw, creating a neuropathic injury. Each created a paw sensitive to external stimulation relative to the contralateral paw. iFU was applied individually to each hind paw in increasing doses until the animal withdrew either paw consistently from the iFU stimulus, thereby defining the iFU threshold dose for that animal. This data was correlated with paw withdrawal latencies to a heat lamp (Hargreaves) test.

Sensitized paws responded to lower intensities and doses of iFU than control paws greater than 95 percent of the time with sensitivities and specificities generally greater than 90 percent. In general, iFU threshold tests and Hargreaves tests did not affect one another, a functional test of the safety of iFU. This preliminary evidence supports the hypothesis that iFU can safely discriminate between painful and normal tissue. Future work will use image-guided iFU to localize deep rather than superficial pain generators.

Delivery of plasmid DNA can be enhanced by treatment with ultrasound (US); acoustic cavitation appears to play an important role in the process. Ultrasound contrast agents (UCAs; stabilized microbubbles) nucleate acoustic cavitation, and lower the acoustic pressure threshold for inertial cavitation occurrence. Fifty micrograms of a liver-specific, high-expressing human factor IX plasmid, pBS-HCRHP-FIXIA, mixed with UCA or phosphate-buffered saline was delivered to mouse livers by intrahepatic injection, with simultaneous exposure to 1 MHz-pulsed US using various acoustic protocols. Variable pulse duration (PD) at constant treatment time, pulse repetition frequency, and an acoustic peak negative pressure amplitude of 1.8 MPa produced 2- to 13-fold enhancements in hFIX gene expression, but PD was not a strong determinant.

In contrast, a dose–response relationship was demonstrated for the peak negative pressure (P ^—), with significant enhancement of gene transduction at P ^— ≥ 2 MPa. Up to 63 ng/ml (approaching the therapeutic range for treating hemophilia patients) could be achieved by transducing one liver lobe at 4-MPa P ^—, corresponding to a 66- fold increment relative to treatment with naked DNA alone. Under the same conditions, mouse livers could also be transduced with a GFP plasmid. Histology showed transient liver damage caused by intrahepatic injection and US exposure at 4-MPa P ^—; however, the damage was repaired in a few days. We conclude that therapeutic US in combination with UCA has the potential to promote safe and efficient nonviral gene transfer of hFIX for the treatment of hemophilia.

This study was performed to identify the affect of variations in the surface activity of the polymer poly(propylacrylic acid) (PPAA) on cavitation-induced hemolysis. The surface activity of PPAA was varied by changing the molecular weight (MW₁ = 43 kDa and MW₂ = 60 kDa) and the solution pH (pH = 5.0, 6.1, and 7.4). Acoustic energy was delivered with a 1.1-MHz high-intensity focused ultrasound transducer. Comparing the two molecular weights, the 60-kDa polymer was a better agent for nucleating cavitation independent of the solution pH, and was a better agent for enhancing cavitation-induced hemolysis.

Background
Systemic metastasis of glioblastoma multiforme (GBM) in the form of bulk tumor is rare. This could be because of patient death before clinically detectable systemic metastasis, impediments to systemic egress, or the inability of GBM to grow outside the central nervous system (CNS). In the present paper, we tested this last hypothesis.

Methods
The delayed brain tumor (DBT) cell was characterized with respect to in vitro and in vivo morphology, growth rate, anchorage-independent growth, glial fibrillary acidic protein expression and cytogenetic analysis, and major histocompatibility complex (MHC) typing. We then assayed implantation-induced intracerebral and systemic GBM growth using 3 rodent models with increasing relative immunologic differences between implanted DBT cells and hosts (Balb/c mice, an isograft, MHC I H2, class type D; C3H mice, an allograft; Wistar rats, a xenograft).

Results
After implantation in the brain, DBT cells generated tumors that were similar to human GBM. Intracerebral DBT implantation as an isograft or allograft produced only intracranial tumors, whereas intracerebral and systemic implantation as a xenograft produced no tumors. Systemic isograft implantation yielded only systemic tumors. Systemic implantation as allografts produced only transient subcutaneous masses.

Conclusions
Delayed brain tumor cells implanted outside the CNS formed tumors unless there was a significant difference between the immunotype of the implanted cells and host. These results support the hypothesis that the rarity of systemic GBM tumors lies in the presence of physical barriers and/or systemic hurdles that prevent their timely growth. These results also demonstrate that GBMs are antigenic, although not immunogenic, with their syngeneic host. Therefore, GBM may be amenable to targeted immunotherapy given successful artificial priming of the immune system.

The aim of this study was the quantitative assessment of the time course and spatial distribution in brain of invading glioblastoma (GBM) cells using a recently described model consisting of RT2 rat GBM cells stably transfected with enhanced green fluorescent protein (eGFP) — called RT2-eGFP — and implanted in Fischer rats. Invasion throughout the brain was verified by confocal microscopy and immunocytochemical staining for eGFP. Rats were sacrificed on post-implantation days 3, 8, and time of death (TOD). First, the entire rat brain was disaggregated at each time point and viable RT2-eGFP cells were counted using flow cytometry with fluorescence as the marker. Next, 2 mm³ samples of cortex from each of four brain quadrants (bifrontal and bioccipital) were disaggregated at each time point, with tumor cell quantification as before. Tumor cell density, averaged over the entire brain, reached a peak mid-way through its time course, leveling out by TOD. Tumor cell density within bulk tumor (BT) was greatest early in the evolution of the brain tumor, decreasing to its final value mid-way through its time course, due to necrosis. The greatest concentration of tumor cells was within BT, with up to an order of magnitude fewer cells in the periphery, while the number of brain tumor cells invading brain distant from BT remained constant from day 3 until TOD. BT size steadily increased after implantation, with an increasing portion due to central necrosis as time progressed, suggesting that this effect is an important contributor to fatality in this model. Alternatively (or additionally), accumulation of toxins elaborated by tumor cells throughout the brain starting early in the evolution of the tumor may also contribute to fatality.

Letter to the Editor

High-intensity focused ultrasound (HIFU) has been shown to generate lesions that destroy brain tissue while disrupting the blood-brain barrier (BBB) in the periphery of the lesion. BBB opening, however, has not been shown without damage, and the mechanisms by which HIFU induces BBB disruption remain unknown. We show that HIFU is capable of reversible, nondestructive, BBB disruption in a targeted region-of-interest (ROI) (29 of 55 applications; 26 of 55 applications showed no effect); this opening reverses after 72 h. Light microscopy demonstrates that HIFU either entirely preserves brain architecture while opening the BBB (18 of 29 applications), or generates tissue damage in a small volume within the region of BBB opening (11 of 29 applications). Electron microscopy supports these observations and suggests that HIFU disrupts the BBB by opening capillary endothelial cell tight junctions, an isolated ultrastructural effect that is different from the mechanisms through which other (untargeted) modalities, such as hyperosmotic solutions, hyperthermia and percussive injury disrupt the BBB.

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/cm². 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 (r² = 0.11) between aggregation and ultrasound intensity, but a substantial correlation (r² = 0.76) between aggregation and cavitation occurrence.

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.

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.

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.

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.

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.

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.

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(R) (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(R) II (Coulter Diagnostics, Miami, FL) and whole blood solutions, showed that P1 increased with increasing Albunex(R) concentration and decreased with increasing PRF, whereas P2 decreased with increasing Albunex(R) 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(R) 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.

Using platelet-rich plasma, we investigated the capability of 1.1-MHz cw high-intensity focused ultrasound (HIFU) to produce "acoustic primary hemostasis," including platelet activation, aggregation, and adhesion to a collagen-coated surface. Platelet activity was evaluated for exposure durations of 100–500 s at intensities of 0–2250 W/cm². In order to avoid heating effects, temperatures in platelet trials were maintained below 42°C through use of a tank cooling system and control of exposure parameters. Flow cytometry, laser aggregometry, conventional microscopy, environmental scanning electron microscopy, and passive cavitation detection were used to quantify platelet activation, aggregation, adhesion, and associated cavitation. HIFU can activate platelets and cause them to adhere to a collagen-coated surface. Cavitation was monitored 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 weak correlation between aggregation and intensity, and a strong correlation between aggregation and cavitation occurrence.

Ultrasound has been used for decades as a means for noninvasive treatment of diseases. Low-intensity ultrasound is routinely applied in physical therapy for muscular and neurological related illnesses. In contrast, high-intensity focused ultrasound (HIFU) is used to induce coagulative necrosis of tissue for cancer treatment or hemostasis. Our efforts concern the latter. Predictions of ultrasound fields, temperature response, and lesion dynamics are obtained by a model which accounts for nonlinear sound propagation in inhomogeneous media, an arbitrary frequency power law for acoustic attenuation, and temperature time history [J. Acoust. Soc. Am. 107, No. 5, Pt. 2 (2000)]. The model is expanded from its previous version to include attenuation and sound speed dependence on temperature levels and also to consider generation of gas bubbles within the tissue. Results are presented in terms of treatment strategies that provide maximum energy transfer for coagulating the targeted tissue while minimizing damage to the surrounding area.

High intensity focused ultrasound (HIFU) is being considered as a noninvasive method to halt internal bleeding, thus we investigated the capability of HIFU to produce "acoustic primary hemostasis", including platelet activation, aggregation and adhesion to a collagen-coated surface. Various HIFU doses were applied to platelet rich plasma (PRP) with and without ultrasound contrast agents. Flow cytometry, laser aggregometry, environmental scanning electron microscopy and passive cavitation detection were used to quantify platelet activation, aggregation, adhesion and associated cavitation. HIFU can activate platelets and cause them to adhere to a collagen-coated surface. Cavitation was monitored 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 weak correlation between aggregation and intensity, and a strong correlation between aggregation and cavitation occurrence.

Unique simultaneous field observations of wind speed and images of vertically polarized radar backscatter from the ocean surface at low grazing angles during low wind and weakly stable stratification show interesting similarities and an important difference, depending on whether the average wind is falling or rising. When the wind speed begins to fall, there is a delay before the area-averaged radar backscatter begins to fall. When the wind speed begins to rise, the backscatter rises immediately. This observation points to hysteresis in the relationship between radar backscatter and wind speed such that more wind is necessary to generate radar backscatter of a given intensity than is necessary to maintain it. This likely arises from differences between the generation of wind-driven gravity capillary waves and the maintenance of gravity capillary waves and/or microbores. Also, saturation was observed in the radar backscatter at wind speeds between 2.1 and 2.8 m s^-1 for both rising and falling winds. This saturation may be related to the energetics of the centimeter-scale gravity capillary waves and/or microbores that cause the backscatter. During rising winds the backscatter may saturate because fine-scale features of the ocean surface reach a maximum amplitude well before wind-derived energy is transferred upscale to larger waves, whose tilting effect would ultimately produce the familiar increase in radar backscatter with increasing wind. During falling winds the backscatter may saturate because of residual momentum in the surface wave field. Finally, for both rising and falling winds, radar backscatter and wind speed are, in general, correlated above a wind speed of 1.7 m s^-1, while below this value they are not. This observation of the critical wind speed necessary to generate or maintain wind-related backscatter is consistent with results from a recent field observation and with existing laboratory-based observations.

Synthetic aperture radar (SAR) has proven to be a useful tool for observing a wide variety of oceanographic and atmospheric phenomena. This is because capillary waves whose amplitudes are modulated in space and time by oceanic and atmospheric processes are efficient scatterers at SAR wavelengths. In this paper, a SAR image of Lake Michigan taken during the Lake-Induced Convection Experiment is analyzed. The image shows three broad parallel bands identifiable as the components of a shallow, Great Lake-induced thermal circulation:two bands associated with opposing land-breeze circulations, and a middle band containing the signature of boundary layer convection. A cross-frontal cut shows that the width of the two land-breeze fronts varies in a manner consistent with previously reported observations of land and sea breezes superimposed on synoptic flows. The SAR image analysis in conjunction with a mesoscale analysis of a Great Lake-scale convection pattern substantially increases the available knowledge of that pattern. Specifically, the SAR image provides information concerning the precise placement of the surface land-breeze fronts not available from other means. Finally, the SAR analysis shows that the western land-breeze brightness patterns are affected by the shallow terrain along the western shore of Lake Michigan. The latter point therefore suggests that SAR can provide valuable information about the link between variations in surface roughness and/or land use patterns and the horizontal structure of the surface wind stress over coastal regions.

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.

In order to simulate ultrasound propagation and subsequent thermal effects in biological media in which blood vessels and other structures may be present, a three-dimensional model has been developed that eliminates the need for symmetry constraints. The model is based on the coupled solution of the full wave nonlinear equation of sound in a lossy medium and the bioheat equation obtained by a pseudospectral finite-difference method in the time domain. It includes nonlinear sound propagation, an arbitrary frequency power law for attenuation, and is capable of treating material inhomogeneities. Unlike other models based on parabolic approximations, it is not restricted to near-axis solutions and can account for reflections and backscattered fields. The program was used to simulate the application of high-intensity focused ultrasound (HIFU) in liver with a blood vessel placed perpendicular to the axis of the transducer and near the focus. This approach follows recent work by the authors [Curra et al., IEEE Trans. Ultrason. Ferroelectr., Freq. Control (in press)]. Simulations are presented for different levels of driving pressure, sound nonlinearities, exposure times, and the relative position between the transducer focus and the blood vessel.

High-intensity focused ultrasound (HIFU) has been examined as a noninvasive means for achieving acoustic hemostasis [Delon–Martin et al., UMB 21, 113-119 (1995); Hynynen et al., UMB 22, 193-201 (1996); Vaezy et al., UMB 24, 903-910 (1998)]. Our own efforts in acoustic hemostasis are directed toward using diagnostic ultrasound to locate a hemorrhage and HIFU to halt the bleeding. To enhance the imaging of blood, the use of ultrasound contrast agents (UCAs; gas-filled microbubbles that increase the echogenicity of fluids) has been proposed as a means to locate internal bleeding; however, the combination of UCAs and ultrasound has been found to cause bioeffects in whole blood [Miller et al., UMB 23, 625-633 (1997); Poliachik et al., UMB 25, 991-998 (1999)]. Our results have shown that HIFU can cause platelets in a platelet rich plasma (PRP) sample to activate, aggregate, and adhere to a collagen-coated surface. Furthermore, UCAs can increase the amount of cavitation induced by HIFU, and thus lead to an increase in platelet activity. Although HIFU exposure alone can induce platelet activity, the addition of UCAs increases the amount and the rate of cavitation (cavitation dose); therefore, cavitation is the likely mechanism of HIFU-induced platelet activity.

The effect of numerous stimuli upon the release of drugs and other molecules from polymeric substrates has been of great interest in the drug delivery community. Ultrasound has proven to be an effective stimulus for the controlled release of drugs from polymer films. However, the mechanism by which this controlled response occurs has yet to be fully understood. In this study, the ability of shear forces generated by microstreaming around a single ultrasonically stimulated bubble to reversibly increase the release of the drugs from a coated polymer film is demonstrated. The polymer film is loaded with the drug ciprofloxacin and then coated with methylene chains consisting of 12 hydrocarbon chains. The leaching rate of the drug thus depends upon the extent of surface coverage by the methylene chains. A single oscillating bubble in a fluid medium has the capacity to drive streaming at the surface of the polymer film and disrupt the methylene chain coating. An increase of drug concentration in suspension as high as ten times the baseline and controls was achieved, which implies an increase in the leaching rate. After treatment, the leaching rate returns to baseline levels, suggesting the methylene chains reorganize upon the polymer surface.

Ultrasound contrast agents have proven to be an effective agent for producing cavitation-associated bioeffects. A comparative analysis of the strength and duration of cavitation activity and the lifetime of nucleation sites during red blood cell (RBC) lysis and sonoporation was performed. The RBCs were treated with 1.1-MHz tone-burst ultrasound in the presence of contrast agents and analyzed for hemoglobin release and the uptake of FITC-0dextran (MW= 4 kD). Pulse durations ranged from 100 to 10,000 ms and the total number of bursts for each pulse duration was selected to ensure a constant exposure. The extent and duration of cavitation activity during treatment was monitored using a passive cavitation detection system. The collected cavitation-activity data was analyzed quantitatively and correlated with the percent of hemolysis and the ratio of fluorescent cells to total cells treated.

There has been some research in the late 1980s on the development and use of biologically compatable, drug-carrying polymers for the purpose of subcutaneous release of the drug. In those papers, ultrasound was used to release the drug. This intriguing method for temporally targeted drug release has been hampered by a large, non-ultrasonic drug release rate. In other words, even without the application of ultrasonic stimulation, an excessive amount of the drug leaches out of the drug-carrying implant. We have developed a bio-compatable, drug-carrying polymer that can release a drug of interest upon ultrasonic stimulation, but whose background leaching rate is negligible. The advance in the present study over previous work is the coating of the drug-carrying polymer with a self-assembling membrane (SAM). Before and after the application of ultrasound, the SAM acts as an effective barrier. During and shortly after the application of ultrasound, the SAM transiently disassembles — thereby releasing the drug — then reassembles, which cuts off the drug flux. We present in vitro results demonstrating this effect, with insulin and ciprofloxin as candidate drugs.

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°C. Unpolarized Fourier transform infrared-attenuated total reflection showed that, after 30 min, the stretching frequencies, vCH_2,asym and vCH_2,sym, decreased and approached 2920 and 2850 cm^-1, indicative of a crystalline phase. The distance between two hydroxyl groups is roughly 4 Å. A tilt angle of 33.5° ± 2.4° was estimated by dichroic 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 severe 5-day lake-effect storm resulted in eight deaths, hundreds of injuries, and over $3 million in damage to a small area of northeastern Ohio and northwestern Pennsylvania in November 1996. In 1999, a blizzard associated with an intense cyclone disabled Chicago and much of the U.S. Midwest with 30–90 cm of snow. Such winter weather conditions have many impacts on the lives and property of people throughout much of North America. Each of these events is the culmination of a complex interaction between synoptic-scale, mesoscale, and microscale processes.

An understanding of how the multiple size scales and timescales interact is critical to improving forecasting of these severe winter weather events. The Lake-Induced Convection Experiment (Lake-ICE) and the Snowband Dynamics Project (SNOWBAND) collected comprehensive datasets on processes involved in lake-effect snowstorms and snowbands associated with cyclones during the winter of 1997/98. This paper outlines the goals and operations of these collaborative projects. Preliminary findings are given with illustrative examples of new state-of-the-art research observations collected. Analyses associated with Lake-ICE and SNOWBAND hold the promise of greatly improving our scientific understanding of processes involved in these important wintertime phenomena.

Work is described whose long-term goal is the extraction and analysis of fine-scale wind fields from synthetic aperture radar (SAR) images of the ocean surface. Results are based on a field experiment in which a SAR image was taken nearly simultaneously with in situ aircraft-based turbulence and radar altimeter measurements. These results show a direct correlation between SAR "streaks" and atmospheric roll vortices. They also show that rolls generate mesoscale variability in the surface wave spectrum, implying that SAR streaks are due to ocean surface roughness elements created by both the instantaneous and time-averaged multiscale wind field. We are using these results to refine an analysis aimed at transforming radar backscatter into fine-scale marine mean wind speed.