Sandra L. Poliachik

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.

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.

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(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.

Control of splenic bleeding by using high intensity ultrasound.

BACKGROUND High-intensity focused ultrasound (HIFU) has been shown to control bleeding from liver incisions, and blood vessel punctures and incisions. The objective of the current study was to investigate the capability of HIFU to stop bleeding from splenic injuries in a pig model. METHODS Surgical incisions, 25 to 50 mm in length and 2 to 8 mm in depth, were made in the spleens of five anesthetized pigs. HIFU with a frequency of 5 MHz was applied within 5 seconds of making the incision. A total of 39 incisions and HIFU treatments were performed. RESULTS Bleeding from all incisions was stopped completely after HIFU treatment. The average times to control and completely arrest the hemorrhage were 28 and 55 seconds, respectively. The mechanisms of hemostasis appeared to be thermally induced coagulation necrosis of splenic tissue and occlusion of blood vessels by a mechanically induced homogenized splenic tissue. CONCLUSION HIFU may provide a useful method of hemostasis for actively bleeding spleen. Because of its ability to induce hemostasis at adjustable depth, HIFU may prove to be a useful cauterization method both in the operating room and for patients who are managed nonoperatively.

RAD001 (Everolimus) inhibits growth of prostate cancer in the bone and the inhibitory effects are increased by combination with docetaxel and zoledronic acid

mTOR activity is increased in advanced prostate cancer (CaP) as a result of a high rate of PTEN mutations. RAD001 (Everolimus) is a new orally available mTOR inhibitor. The objective of our study was to evaluate the effects of RAD001 on the growth of CaP in the bone, both alone and in combination with docetaxel and zoledronic acid.

Association of MTOR Mutations With Developmental Brain Disorders, Including Megalencephaly, Focal Cortical Dysplasia, and Pigmentary Mosaicism

IMPORTANCE Focal cortical dysplasia (FCD), hemimegalencephaly, and megalencephaly constitute a spectrum of malformations of cortical development with shared neuropathologic features. These disorders are associated with significant childhood morbidity and mortality. OBJECTIVE To identify the underlying molecular cause of FCD, hemimegalencephaly, and diffuse megalencephaly. DESIGN, SETTING, AND PARTICIPANTS Patients with FCD, hemimegalencephaly, or megalencephaly (mean age, 11.7 years; range, 2-32 years) were recruited from Pediatric Hospital A. Meyer, the University of Hong Kong, and Seattle Childrens Research Institute from June 2012 to June 2014. Whole-exome sequencing (WES) was performed on 8 children with FCD or hemimegalencephaly using standard-depth (50-60X) sequencing in peripheral samples (blood, saliva, or skin) from the affected child and their parents and deep (150-180X) sequencing in affected brain tissue. Targeted sequencing and WES were used to screen 93 children with molecularly unexplained diffuse or focal brain overgrowth. Histopathologic and functional assays of phosphatidylinositol 3-kinase-AKT (serine/threonine kinase)-mammalian target of rapamycin (mTOR) pathway activity in resected brain tissue and cultured neurons were performed to validate mutations. MAIN OUTCOMES AND MEASURES Whole-exome sequencing and targeted sequencing identified variants associated with this spectrum of developmental brain disorders. RESULTS Low-level mosaic mutations of MTOR were identified in brain tissue in 4 children with FCD type 2a with alternative allele fractions ranging from 0.012 to 0.086. Intermediate-level mosaic mutation of MTOR (p.Thr1977Ile) was also identified in 3 unrelated children with diffuse megalencephaly and pigmentary mosaicism in skin. Finally, a constitutional de novo mutation of MTOR (p.Glu1799Lys) was identified in 3 unrelated children with diffuse megalencephaly and intellectual disability. Molecular and functional analysis in 2 children with FCD2a from whom multiple affected brain tissue samples were available revealed a mutation gradient with an epicenter in the most epileptogenic area. When expressed in cultured neurons, all MTOR mutations identified here drive constitutive activation of mTOR complex 1 and enlarged neuronal size. CONCLUSIONS AND RELEVANCE In this study, mutations of MTOR were associated with a spectrum of brain overgrowth phenotypes extending from FCD type 2a to diffuse megalencephaly, distinguished by different mutations and levels of mosaicism. These mutations may be sufficient to cause cellular hypertrophy in cultured neurons and may provide a demonstration of the pattern of mosaicism in brain and substantiate the link between mosaic mutations of MTOR and pigmentary mosaicism in skin.

Transient muscle paralysis disrupts bone homeostasis by rapid degradation of bone morphology

We have previously shown that transient paralysis of murine hindlimb muscles causes profound degradation of both trabecular and cortical bone in the adjacent skeleton within 3 weeks. Morphologically, the acute loss of bone tissue appeared to arise primarily due to osteoclastic bone resorption. Given that the loss of muscle function in this model is transient, we speculated that the stimulus for osteoclastic activation would be rapid and morphologic evidence of bone resorption would appear before 21 days. We therefore utilized high-resolution in vivo serial micro-CT to assess longitudinal alterations in lower hindlimb muscle volume, proximal tibia trabecular, and tibia mid-diaphysis cortical bone morphology in 16-week-old female C57 mice following transient calf paralysis from a single injection of botulinum toxin A (BtA; 2U/100 g body weight). In an acute study, we evaluated muscle and bone alterations at days 0, 3, 5, and 12 following transient calf paralysis. In a chronic study, following day 0 imaging, we assessed the recovery of these tissues following the maximum observed trabecular degradation (day 12) through day 84 post-paralysis. The time course and degree of recovery of muscle, trabecular, and cortical bone varied substantially. Significant atrophy of lower limb muscle was evident by day 5 of paralysis, maximal at day 28 (-34.1+/-0.9%) and partially recovered by day 84. Trabecular degradation within the proximal tibia metaphysis occurred more rapidly, with significant reduction in BV/TV by day 3, maximal loss at day 12 (-76.8+/-2.9%) with only limited recovery by day 84 (-51.7+/-5.1% vs. day 0). Significant cortical bone volume degradation at the tibia mid-diaphysis was first identified at day 12, was maximal at day 28 (-9.6+/-1.2%), but completely recovered by day 84. The timing, magnitude, and morphology of the observed bone erosion induced by transient muscle paralysis were consistent with a rapid recruitment and prolific activation of osteoclastic resorption. In a broader context, understanding how brief paralysis of a single muscle group can precipitate such rapid and profound bone resorption in an adjacent bone is likely to provide new insight into how normal muscle function modulates bone homeostasis.

Multimodality localization of the sensorimotor cortex in pediatric patients undergoing epilepsy surgery

OBJECT The gold-standard method for determining cortical functional organization in the context of neurosurgical intervention is electrical cortical stimulation (ECS), which disrupts normal cortical function to evoke movement. This technique is imprecise, however, as motor responses are not limited to the precentral gyrus. Electrical cortical stimulation also can trigger seizures, is not always tolerated, and is often unsuccessful, especially in children. Alternatively, endogenous motor and sensory signals can be mapped by somatosensory evoked potentials (SSEPs), functional MRI (fMRI), and electrocorticography of high gamma (70-150 Hz) signal power, which reflect normal cortical function. The authors evaluated whether these 4 modalities of mapping sensorimotor function in children produce concurrent results. METHODS The authors retrospectively examined the charts of all patients who underwent epilepsy surgery at Seattle Childrens Hospital between July 20, 1999, and July 1, 2011, and they included all patients in whom the primary motor or somatosensory cortex was localized via 2 or more of the following tests: ECS, SSEP, fMRI, or high gamma electrocorticography (hgECoG). RESULTS Inclusion criteria were met by 50 patients, whose mean age at operation was 10.6 years. The youngest patient who underwent hgECoG mapping was 2 years and 10 months old, which is younger than any patient reported on in the literature. The authors localized the putative sensorimotor cortex most often with hgECoG, followed by SSEP and fMRI; ECS was most likely to fail to localize the sensorimotor cortex. CONCLUSIONS Electrical cortical stimulation, SSEP, fMRI, and hgECoG generally produced concordant localization of motor and sensory function in children. When attempting to localize the sensorimotor cortex in children, hgECoG was more likely to produce results, was faster, safer, and did not require cooperation. The hgECoG maps in pediatric patients are similar to those in adult patients published in the literature. The sensorimotor cortex can be mapped by hgECoG and fMRI in children younger than 3 years old to localize cortical function.

The magnetic resonance imaging spectrum of facioscapulohumeral muscular dystrophy

Introduction: Facioscapulohumeral muscular dystrophy (FSHD) is associated with a repeat contraction in the D4Z4 gene locus on chromosome 4q35. We used a one‐step quantitative magnetic resonance imaging (MRI) method to evaluate muscle, edema, and fat in patients spanning the range of severity. Methods: Fifteen patients with FSHD were compared with 10 healthy subjects using non‐negative linear least‐squares fitting of 32‐echo relaxation data (T2). The results were compared with a biexponential approach for characterizing muscle/fat ratio and T2 relaxation measurements from fat‐suppressed inversion recovery. Results: Increased T2 signal consistent with edema was common in FSHD subjects, a pattern not present in healthy controls. A varied pattern of edema and fatty replacement in muscles was shown. Conclusions: As a discrete biomarker, edema may be useful for following the clinical course of FSHD. Future work toward optimizing measurement is discussed. Muscle Nerve, 2012

Why Rest Stimulates Bone Formation: A Hypothesis Based on Complex Adaptive Phenomenon

GROSS, T. S., S. L. POLIACHIK, B. J. AUSK, D. A. SANFORD, B. A. BECKER, and S. SRINIVASAN. Why rest stimulates bone formation: A hypothesis based on complex adaptive phenomenon. Exerc. Sport Sci. Rev., Vol. 32, No. 1, pp. 9–13, 2004. Moderate exercise is an ineffective strategy to build bone mass. The authors present data demonstrating that allowing bone to rest between each load cycle transforms low- and moderate-magnitude mechanical loading into a signal that potently induces bone accretion. They hypothesize that the osteogenic nature of rest-inserted loading arises by enabling osteocytes to communicate as a small world network.