Evaluation of Thiel cadaveric model for MRI-guided stereotactic procedures in neurosurgery

BACKGROUND: Magnetic resonance imaging (MRI)-guided deep brain stimulation (DBS) and high frequency focused ultrasound (FUS) is an emerging modality to treat several neurological disorders of the brain. Developing reliable models to train and assess future neurosurgeons is paramount to ensure safety and adequate training of neurosurgeons of the future. METHODS: We evaluated the use of Thiel cadaveric model to practice MRI-guided DBS implantation and high frequency MRI-guided FUS in the human brain. We performed three training sessions for DBS and five sonications using high frequency MRI-guided FUS in five consecutive cadavers to assess the suitability of this model to use in training for stereotactic functional procedures. RESULTS: We found the brains of these cadavers preserved in an excellent anatomical condition up to 15 months after embalmment and they were excellent model to use, MRI-guided DBS implantation and FUS produced the desired lesions accurately and precisely in these cadaveric brains. CONCLUSION: Thiel cadavers provided a very good model to perform these procedures and a potential model to train and assess neurosurgeons of the future

How sonoporation disrupts cellular structural integrity: morphological and cytoskeletal observations

Posters: no. 1Control ID: 1672429OBJECTIVES: In considering sonoporation for drug delivery applications, it is essential to understand how living cells respond to this puncturing force. Here we seek to investigate the effects of sonoporation on cellular structural integrity. We hypothesize that the membrane morphology and cytoskeletal behavior of sonoporated cells under recovery would inherently differ from that of normal viable cells. METHODS: A customized and calibrated exposure platform was developed for this work, and the ZR-75-30 breast carcinoma cells were used as the cell model. The cells were exposed to either single or multiple pulses of 1 MHz ultrasound (pulse length: 30 or 100 cycles; PRF: 1kHz; duration: up to 60s) with 0.45 MPa spatial-averaged peak negative pressure and in the presence of lipid-shelled microbubbles. Confocal microscopy was used to examine insitu the structural integrity of sonoporated cells (identified as ones with exogenous fluorescent marker internalization). For investigations on membrane morphology, FM 4-64 was used as the membrane dye (red), and calcein was used as the sonoporation marker (green); for studies on cytoskeletal behavior, CellLight (green) and propidium iodide (red) were used to respectively label actin filaments and sonoporated cells. Observation started from before exposure to up to 2 h after exposure, and confocal images were acquired at real-time frame rates. Cellular structural features and their temporal kinetics were quantitatively analyzed to assess the consistency of trends amongst a group of cells. RESULTS: Sonoporated cells exhibited membrane shrinkage (decreased by 61% in a cell’s cross-sectional area) and intracellular lipid accumulation (381% increase compared to control) over a 2 h period. The morphological repression of sonoporated cells was also found to correspond with post-sonoporation cytoskeletal processes: actin depolymerization was observed as soon as pores were induced on the membrane. These results show that cellular structural integrity is indeed disrupted over the course of sonoporation. CONCLUSIONS: Our investigation shows that the biophysical impact of sonoporation is by no means limited to the induction of membrane pores: e.g. structural integrity is concomitantly affected in the process. This prompts the need for further fundamental studies to unravel the complex sequence of biological events involved in sonoporation.postprin

Developmental delays and subcellular stress as downstream effects of sonoporation

Posters: no. 2Control ID: 1672434OBJECTIVES: The biological impact of sonoporation has often been overlooked. Here we seek to obtain insight into the cytotoxic impact of sonoporation by gaining new perspectives on anti-proliferative characteristics that may emerge within sonoporated cells. We particularly focused on investigating the cell-cycle progression kinetics of sonoporated cells and identifying organelles that may be stressed in the recovery process. METHODS: In line with recommendations on exposure hardware design, an immersion-based ultrasound platform has been developed. It delivers 1 MHz ultrasound pulses (100 cycles; 1 kHz PRF; 60 s total duration) with 0.45 MPa peak negative pressure to a cell chamber that housed HL-60 leukemia cells and lipid-shelled microbubbles at a 10:1 cell-tobubble ratio (for 1e6/ml cell density). Calcein was used to facilitate tracking of sonoporated cells with enhanced uptake of exogenous molecules. The developmental trend of sonoporated cells was quantitatively analyzed using BrdU/DNA flow cytometry that monitors the cell population’s DNA synthesis kinetics. This allowed us to measure the temporal progression of DNA synthesis of sonoporated cells. To investigate whether sonoporation would upset subcellular homeostasis, post-exposure cell samples were also assayed for various proteins using Western blot analysis. Analysis focus was placed on the endoplasmic reticulum (ER): an important organelle with multi-faceted role in cellular functioning. The post-exposure observation time spanned between 0-24 h. RESULTS: Despite maintaining viability, sonoporated cells were found to exhibit delays in cell-cycle progression. Specifically, their DNA synthesis time was lengthened substantially (for HL-60 cells: 8.7 h for control vs 13.4 h for the sonoporated group). This indicates that sonoporated cells were under stress: a phenomenon that is supported by our Western blot assays showing upregulation of ER-resident enzymes (PDI, Ero1), ER stress sensors (PERK, IRE1), and ER-triggered pro-apoptotic signals (CHOP, JNK). CONCLUSIONS: Sonoporation, whilst being able to facilitate internalization of exogenous molecules, may inadvertently elicit a cellular stress response. These findings seem to echo recent calls for reconsideration of efficiency issues in sonoporation-mediated drug delivery. Further efforts would be necessary to improve the efficiency of sonoporation-based biomedical applications where cell death is not desirable.postprin

A study on the change in plasma membrane potential during sonoporation

Posters: no. 4Control ID: 1680329OBJECTIVES: There has been validated that the correlation of sonoporation with calcium transients is generated by ultrasound-mediated microbubbles activity. Besides calcium, other ionic flows are likely involved in sonoporation. Our hypothesis is the cell electrophysiological properties are related to the intracellular delivery by ultrasound and microbubbles. In this study, a real-time live cell imaging platform is used to determine whether plasma membrane potential change is related to the sonoporation process at the cellular level. METHODS: Hela cells were cultured in DMEM supplemented with 10% FBS in Opticell Chamber at 37 °C and 5% CO2, and reached 80% confluency before experiments. The Calcein Blue-AM, DiBAC4(3) loaded cells in the Opticell chamber filled with PI solution and Sonovue microbubbles were immerged in a water tank on a inverted fluorescence microscope. Pulsed ultrasound (1MHz freq., 20 cycles, 20Hz PRF, 0.2-0.5MPa PNP) was irradiated at the angle of 45° to the region of interest for 1s.The real-time fluorescence imaging for different probes was acquired by a cooled CCD camera every 20s for 10min. The time-lapse fluorescence images were quantitatively analyzed to evaluate the correlation of cell viability, intracellular delivery with plasma membrane potential change. RESULTS: Our preliminary data showed that the PI fluorescence, which indicated intracellular delivery, was immediately accumulated in cells adjacent to microbubbles after exposure, suggesting that their membranes were damaged by ultrasound-activated microbubbles. However, the fluorescence reached its highest level within 4 to 6 minutes and was unchanged thereafter, indicating the membrane was gradually repaired within this period. Furthermore, using DIBAC4(3), which detected the change in the cell membrane potential, we found that the loss of membrane potential might be associated with intracellular delivery, because the PI fluorescence accumulation was usually accompanied with the change in DIBAC4 (3) fluorescence. CONCLUSIONS: Our study suggests that there may be a linkage between the cell membrane potential change and intracellular delivery mediated by ultrasound and microbubbles. We also suggest that other ionic flows or ion channels may be involved in the cell membrane potential change in sonoporation. Further efforts to explore the cellular mechanism of this phenomenon will improve our understanding of sonoporation.postprin

Real-time imaging of cellular dynamics during low-intensity pulsed ultrasound exposure

Control ID: 1671584Oral Session 5 - Bioeffects of therapeutic ultrasoundOBJECTIVE: Although the therapeutic potential of low-intensity pulsed ultrasound is unquestionable, the wave-matter interactions involved in the process remain to be vaguely characterized. Here we seek to undertake a series of in-situ cellular imaging studies that aim to analyze the mechanical impact of low-intensity pulsed ultrasound on attached fibroblasts from three different aspects: membrane, cytoskeleton, and nucleus. METHODS: Our experimental platform comprised an in-house ultrasound exposure hardware that was coupled to a confocal microscopy system. The waveguided ultrasound beam was geometrically aligned to the microscope’s fieldof-view that corresponds to the center of a polystyrene dish containing fibroblasts. Short ultrasound pulses (5 cycles; 2 kHz PRF) with 0.8 MPa peak acoustic pressure (0.21 W/cm2 SPTA intensity) were delivered over a 10 min period. Live imaging was performed on both membrane (CellMask) and cytoskeleton (actin-GFP, tubulin-RFP) over the entire observation period (up to 30 min after end of exposure). Also, pre- and post-exposure fixed-cell imaging was conducted on the nucleus (Hoechst 33342) and two cytoskeleton components related to stress fibers: F-actin (phalloidin-FITC) and vincullin (Alexa Fluor 647 conjugated). To study whether mechanotransduction was responsible in mediating ultrasound-cell interactions, some experiments were conducted with the addition of gadolinium that blocks stretch-sensitive ion channels. RESULTS: Cell shrinkage was evident over the course of low-intensity pulsed ultrasound exposure. This was accompanied with contraction of actin and tubulin. Also, an increase in central stress fibers was observed at the end of exposure, while the nucleus was found to have decreased in size. Interestingly, after the exposure, a significant rebound in cell volume was observed over a 30 min. period. These effects were not observed in cases with gadolinium blockage of mechanosensitive ion channels. CONCLUSIONS: Our results suggest that low-intensity pulsed ultrasound would transiently induce remodeling of a cell’s membrane and cytoskeleton, and it will lead to repression of nucleus. This indicates that ultrasound after all represents a mechanical stress on cellular membrane. The post-exposure outgrowth phenomenon is also of practical relevance as it may be linked to the stimulatory effects that have been already observed in low-intensity pulsed ultrasound treatments.postprin

Doctor of Philosophy

dissertationFocused ultrasound (FUS) is a promising noninvasive and radiation-free cancer therapy that selectively delivers high-intensity acoustic energy to a small target volume. This dissertation presents original research that improves the speed, safety, and efficacy of FUS therapies under magnetic resonance imaging (MRI) guidance. First, a new adaptive model-predictive controller is presented that leverages the ability of MRI to measure temperature inside the patient at near real-time speeds. The controller uses MR temperature feedback to dynamically derive and update a patient-specific thermal model, and optimizes the treatment based on the model's predictions. Treatment safety is a key element of the controller's design, and it can actively protect healthy tissue from unwanted damage. In vivo and simulation studies indicate the controller can safeguard healthy tissue and accelerate treatments by as much as 50%. Significant tradeoffs exist between treatment speed, and safety, which makes a real-time controller absolutely necessary for carrying out efficient, effective, and safe treatments while also highlighting the importance of continued research into optimal treatment planning. Next, two new methods for performing 3D MR acoustic radiation force imaging (MR-ARFI) are presented. Both techniques measure the tissue displacement induced by short bursts of focused ultrasound, and provide a safe way to visualize the ultrasound beam's location. In some scenarios, ARFI is a necessity for proper targeting since traditional MR thermometry cannot measure temperature in fat. The first technique for performing 3D ARFI introduces a novel unbalanced bipolar motion encoding gradient. The results demonstrate that this technique is safe, and that 3D displacement maps can be attained time-efficiently even in organs that contain fat, such as breast. The second technique measures 3D ARFI simultaneously with temperature monitoring. This method uses a multi-contrast gradient recalled echo sequence which makes multiple readings of the data without increasing scan time. This improves the signal to noise ratio and makes it possible to separate the effects of tissue heating vs displacement. Both of the 3D MR-ARFI techniques complement the presented controllersince proper positioning of the focal spot is critical to achieving fast and safe treatments

Treatment of brain cancer and ischaemic stroke utilising High Intensity Focus Ultrasound (HIFU) guide with MRI

In this thesis high intensity focused ultrasound (HIFU) is utilized for cancer treatment (thermal mode) and treatment of ischaemic stroke (mechanical mode). These two applications were investigated in vitro and in vivo models. MRI was utilized to monitor the lesions created by HIFU either in thermal or cavitation mode in freshly excised lamb brain tissue in vitro, and in rabbit brain in vivo. Additionally, MRI was used to monitor lesions deep in tissue for both in vitro and in vivo exposures. All three MRI sequences used (T1-W FSE, T2-W FSE and FLAIR) were able to detect lesions. Both thermal and bubbly lesions were best monitored using T1-W FSE with excellent contrast, proving the potential of HIFU to treat reliably tumours in the brain. A HIFU system was also used to assist thrombolysis in cooperation with a thrombolytic drug such as recombinant tissue plasminogen activator (rt-PA) in vitro and in vivo. It was shown that higher intensity results to higher volume of dissolved clot, but there is a limit of the intensity to be used in order to avoid heating of the clot and the surrounding tissue. The goal in this study was to achieve temperature elevation not exceeding 1ºC (called safe temperature). It was found that the larger the beam area the larger the dissolved clot volume. Also, the lower the frequency, the larger the volume of the dissolved clot. The results reported herein point to the use of frequency around 0.5 MHz and pulsing to optimize thrombolysis and skull penetration and at the same time avoiding unwanted heating. Finally, an Acrylonitrile Butadiene Styrene (ABS) phantom skull model was developed in order to evaluate the propagation of ultrasound using a single element transducer. The skull model was appropriately designed so that it has the same attenuation as a human skull. It was demonstrated that using a frequency of 0.5 MHz versus 1 MHz, ultrasound propagation through the phantom skull was higher. Therefore, higher frequency has poor skull penetration and a small beam size at the focus, while low frequencies have better skull penetration but with the risk of reaching the unpredictable effect of cavitation. The developed system has proven to successfully create large lesions in the brain and at the same time, these lesions are successfully monitored with excellent contrast using MRI (T1-W FSE) enabling the accurate determination of the margins of these lesions. The results reported in this study point to the use of frequency around 0.5 MHz and pulsing to optimize thrombolysis and skull penetration and at the same time avoiding unwanted heating. For treating tumours located deep in the brain and for dissolving thrombus causing an acute ischaemic stroke, further extensive clinical studies will be needed before this technology is applied to humans

A Bayesian approach for energy-based estimation of acoustic aberrations in high intensity focused ultrasound treatment

High intensity focused ultrasound is a non-invasive method for treatment of diseased tissue that uses a beam of ultrasound to generate heat within a small volume. A common challenge in application of this technique is that heterogeneity of the biological medium can defocus the ultrasound beam. Here we reduce the problem of refocusing the beam to the inverse problem of estimating the acoustic aberration due to the biological tissue from acoustic radiative force imaging data. We solve this inverse problem using a Bayesian framework with a hierarchical prior and solve the inverse problem using a Metropolis-within-Gibbs algorithm. The framework is tested using both synthetic and experimental datasets. We demonstrate that our approach has the ability to estimate the aberrations using small datasets, as little as 32 sonication tests, which can lead to significant speedup in the treatment process. Furthermore, our approach is compatible with a wide range of sonication tests and can be applied to other energy-based measurement techniques

Focused ultrasound-mediated suppression of chemically-induced acute epileptic EEG activity

<p>Abstract</p> <p>Background</p> <p>Epilepsy is a common neurological disorder, which is attributed to uncontrollable abnormal hyper-excitability of neurons. We investigated the feasibility of using low-intensity, pulsed radiation of focused ultrasound (FUS) to non-invasively suppress epileptic activity in an animal model (rat), which was induced by the intraperitonial injection of pentylenetetrazol (PTZ).</p> <p>Results</p> <p>After the onset of induced seizures, FUS was transcranially administered to the brain twice for three minutes each while undergoing electroencephalographic (EEG) monitoring. An air-backed, spherical segment ultrasound transducer (diameter: 6 cm; radius-of-curvature: 7 cm) operating at a fundamental frequency of 690 KHz was used to deliver a train of 0.5 msec-long pulses of sonication at a repetitive rate of 100 Hz to the thalamic areas of the brain. The acoustic intensity (130 mW/cm²) used in the experiment was sufficiently within the range of safety guidelines for the clinical ultrasound imaging. The occurrence of epileptic EEG bursts from epilepsy-induced rats significantly decreased after sonication when it was compared to the pre-sonication epileptic state. The PTZ-induced control group that did not receive any sonication showed a sustained number of epileptic EEG signal bursts. The animals that underwent sonication also showed less severe epileptic behavior, as assessed by the Racine score. Histological analysis confirmed that the sonication did not cause any damage to the brain tissue.</p> <p>Conclusions</p> <p>These results revealed that low-intensity, pulsed FUS sonication suppressed the number of epileptic signal bursts using acute epilepsy model in animal. Due to its non-invasiveness and spatial selectivity, FUS may offer new perspectives for a possible non-invasive treatment of epilepsy.</p