François Tranquart

Characterization of cell membrane response to ultrasound activated microbubbles

Contrast agents for ultrasound imaging, composed of tiny gas microbubbles, have become a reality in clinical routine. They are extensively used in radiology for detection and characterization of various tumors and in cardiology for left ventricular opacification. Recent experimental studies showed that ultrasound waves in combination with contrast agent microbubbles increase transiently cell membrane permeability in a process known as sonoporation. This effect is thought to allow foreign molecules to enter the cell. In that context, we explored the cell membranes responses to microbubbles oscillations as the mechanism is not completely understood. Breast cancer cell line in combination with contrast microbubbles were used. Ultrasound was applied using a transducer of 1 MHz center frequency transmitting a 10-cycle burst of different acoustic pressures repeated every 100 mus. Patch-clamp technique in whole cell configuration was used to explore transmembrane ion exchange through the variations in membrane potential. To characterize the activated ion channels, the variations of the intracellular calcium (Ca2+) concentration were explored using a fluorescent marker. The results revealed that ultrasound stimulation induces a rapid hyperpolarization of cell membrane potential when the microbubble is in direct contact with the cell, but the potential returned to its initial value when ultrasound stimulation stopped. The change in cell membrane potential indicates the activation of specific ion channels and depends on the quality of microbubble adhesion to the cell membrane. Microbubbles were shown to induce a mechanical stretch activating BKca channels. Simultaneous Ca2+ measurements indicate a slow and progressive Ca2+ increase that is likely a consequence of BKca channels opening not a cause. These results demonstrate that microbubbles oscillations under ultrasound activation entail modulation of cellular function and signaling by triggering the modulation of ionic transports through the cell membrane. Cells response to the mechanical stretch caused by gentle microbubble oscillations is characterized by the opening of BKca stretch channels and a Ca2+ flux, which might potentially trigger other cellular responses responsible for membrane sonopermeabilization.

Piezocomposite and CMUT arrays assessment through in vitro imaging performances

Piezoelectric and capacitive micromachined ultrasound transducers (CMUT) are usually measured and compared in regards to acoustic and electro-acoustic performances. This paper is focused on the imaging performances of such transducers and propose a quantitative imaging assessment of B-mode images. In this purpose, fully integrated CMUT and piezocomposite-based probes were manufactured. Transducers were designed with close features (geometries, center frequency, interconnect and packaging) and plug on a clinical ultrasound system with a research interface. Major imaging performances (speckle to noise ratio, resolutions and contrast) of the probes are presented. Despite an environment dedicated to piezoelectric transducers, CMUT probe exhibits comparable image quality as compared to a state-of-the-art piezocomposite probe. Indeed, close resolutions are calculated, field of view is improved in phased-array imaging, and contrast is significantly improved.

Recent advances in gene delivery with ultrasound and microbubbles

Recently, microbubbles used in combination with ultrasound has been proposed as an alternative method for in vitro and in vivo gene delivery. Ultrasound (US) and microbubble assisted delivery (USMD) has great clinical potential based on the fact that ultrasound contrast agents (USCAs) are clinically approved for diagnostic applications. The mechanism that supports gene delivery via USMD is thought to be ‘sonoporation’. It is hypothesised that the interaction of US and USCA can induce a transient cell membrane permeabilisation leading to enhanced DNA uptake. The exact mechanism(s) of gene uptake remains a subject of academic debate and various hypotheses have been proposed, including acoustic microstreaming or local shear stresses, microjetting and cavitation. This review provides the current understanding of USMD and highlights both the bubble characteristics and ultrasound parameters that support USMD in vitro and in vivo. Both safety and efficacy of gene delivery by sonoporation are discussed. In addition, USMD is compared with electroporation, another widely used physical method for gene delivery.

Evaluation of cerebral blood flow in rabbits with transcranial doppler sonography: First results

A transcranial Doppler sonographic system with a special 2-MHz probe for prolonged experimentation has been developed for Doppler waveform recordings from the basilar artery on rabbits. Measurements were made from the maximum velocity wave form, and Pourcelots resistance index was used to express the results. The diastolic flow, similar to that observed in humans in physiological conditions, decreases with increased intracranial pressure during chronic intracranial hypertension. This Doppler model may be valuable for assessing rapid changes of cerebral blood flow in conscious animals during prolonged or acute experimental procedures.

On the Mechanisms of Ultrasound Contrast Agents-Induced Arrhythmias

Contrast ultrasound imaging is a noninvasive imaging modality using gas microbubbles. However disorders of the heart rate expressed by the appearance of premature ventricular contractions (PVCs) occur in few patients. We have recently shown through electrophysiological studies that ultrasound activated microbubbles oscillate and induce a mechanical stress on the cell membrane resulting in the activation of stretch activated channels (SAC). These channels, described initially in the muscular cells, are ionic channels expressed in all cell types. In the heart, their activation can trigger arrhythmias. The aim of this study is to demonstrate, in vivo and in vitro, the relationship between PVC and SAC opening by ultrasound contrast agents (UCA) during ultrasound (US) exposure. Three sets of experiments were conducted: two on animal models with microbubbles and mechanical stress and one in vitro. 5 anesthetized rats were used. PVCs were created in vivo using SonoVuetrade microbubbles at a rate of 0.5 ml/min. The microbubbles were injected intravenously through the tail vein. US consisted of 40-cycle waveforms at 1 MHz and peak negative pressures up to 300 kPa. A PVC animal model based on a manually-induced mechanical stretch was used. The model consisted of stimulations by a flexible catheter introduced into the rat aorta and pushed until the left ventricle. PVCs were quantified through ECG measurements. In-vitro, patch Clamp technique in ruptured-patch whole cell configuration was used to monitor the changes in the electrical potential on HL-1 heart cell line during manual stimulation. In vivo, UCA and US at 300 kPa induced modification of rats ECG while pressure below 300 kPa did not induce any PVC. US alone did not induce PVC at any acoustic pressures. Similar PVCs were also observed in the animal model but ceased immediately after the stimulation. In vitro, electrophysiological recordings showed that the mechanical stretch engendered a cell membrane depolarization due to SAC opening. The cell membrane potential depolarized as a consequence of the manual stimulation and returned to its initial value when the stimulation was released. This effect might trigger additional action potential and by that the generation of extra contractions such as PVCs. We presume that PVCs are triggered through the opening of stretch activated ion channels. This study indicates that PVCs induced by UCA are related to mechanical interaction between cell membrane and oscillating microbubbles.

The DopFet system : A new ultrasonic doppler system for monitoring and characterization of fetal movement

To enable the investigation of fetal movement in a manner similar to fetal heart rate (FHR) monitoring we have developed an apparatus (the DopFet system) that consists of a pair of miniature sensors, a 2-MHz continuous-wave directional Doppler electronic module and a laptop personal computer. One of the sensors is aimed at the fetal limbs and the other at the thorax to detect heart and upper body movements. The signals are analyzed, presented in real-time and postprocessed by software developed by us. The postprocessing software computes a number of parameters (the DopFet parameters) describing fetal movement. These parameters can be divided into two categories: parameters that describe the quantity of fetal movement (i.e., number of movements) and parameters that describe qualitative aspects of fetal movement (i.e., average movement duration). Future studies using the DopFet system will be aimed at discovering which of these parameters or combination of parameters is the best indicator of fetal well-being. We present an example of a 0.5 h recording and the results of testing on 23 volunteer mothers. These results show good sensitivity of the system compared to real-time ultrasound (US). The system detects 96% of rolling movements, 100% of flexion movements and 97% of leg movements.

Detection and analysis of fetal movements by ultrasonic multi-sensor Doppler (ACTIFOETUS)

The major objective for fetal ultrasonic monitoring of pregnancies is to recognize pathological conditions with sufficient warning to enable intervention by the clinician before the occurrence of irreversible fetal lesions. For that purpose we developed a multi detector and multigate Doppler system for the study of fetal movements and fetal rhythms in high risk pregnancies. Three methods of signal and data processing have been evaluated for the analysis of ultrasonic Doppler signals generated by the movements of fetal structures: fast Fourier transform (FFT) autocorrelation function (ACF) and independent component analysis (ICA). Preliminary results obtained in 15 normal pregnancies (32-35 weeks) are shown. Recording duration for each examination was between 15 and 20 minutes.

FIBRINOLYTIC EFFECTS OF TRANSPARIETAL ULTRASOUND ASSOCIATED WITH INTRAVENOUS INFUSION OF AN ULTRASOUND CONTRAST AGENT: STUDY OF A RAT MODEL OF ACUTE CEREBRAL STROKE

The aim of this study was to evaluate the thrombolytic effect of focused transparietal ultrasound in combination with a specific contrast agent (microbubbles) in acute cerebral ischemia. Acute cerebral ischemia was induced in 10 rats by intra-arterial clots injection. Five rats (group 1) were treated with a combination of transparietal ultrasound (probe 2 MHz, acoustic power 500 mW/cm(2)) and intravenous injection of 0.6 mL of the ultrasound contrast agent (UCA) sulfur hexafluoride. Five rats (group 2) were treated by fibrinolytic intravenous infusion (recombinant tissue plasminogen activator). Cerebral cellular energy production was determined by measuring the cellular phosphorylation using phosphorus magnetic spectroscopy before and during ischemia induction and after treatment. Measures were performed on a dedicated 2.35T magnet. The ratio phosphocreatine (P(Cr)) on inorganic phosphate (P(i)), P(Cr)/P(i), estimation of the oxidative phosphorylation metabolism and the intracellular pH (pHi) were measured in the two groups. Compared with the ischemia induction period, both treatments were associated with an increase of P(Cr)/P(i) and pHi values, respectively, +80% and +100% in group 1 (p=0.07) and +100% and +80% in group 2 (p=0.04). There was no significant difference between the two groups for the response treatment. To conclude, treatment with intravenous fibrinolytic infusion and treatment with focused ultrasound in combination with UCA seems to be equally effective in treating acute cerebral ischemia in rats. (E-mail: j.p.tasu@chu-poitiers.fr).

Synchronous Detection of Emboli by wavelet packet decomposition

We showed in a previous study that the detection of micro-emboli can be markedly improved by taking into account the quasi-cyclostationary properties of the blood Doppler signal. However, in order to detect still smaller events, we propose to combine the use of synchronous detectors and sub-band decomposition. We evaluated and compared the detectors performance to existing methods by using both numerical modeling of the Doppler signal and ROC curves.

Automatic lesions segmentation in ultrasound nonlinear imaging

Doppler has been used for many years for cardiovascular exploration in order to visualize the vessels walls and anatomical or functional diseases. The use of ultrasound contrast agents makes it possible to improve ultrasonic information. Nonlinear ultrasound imaging highlights the detection of these agents within an organ and hence is a powerful technique to image perfusion of an organ in real-time. The visualization of flow and perfusion provides important information for the diagnosis of various diseases as well as for the detection of tumors. We present a new method to automatically segment hepatic tumors, during a perfusion phase, in ultrasound hepatic nonlinear imaging. It is based on a coherence enhancing diffusion process, which removes noise and raises the structural characteristics. The segmentation procedure is a fuzzy competitive agglomerative clustering which takes into account of the perfusion phenomenon. We have tested our method on clinical sequences and obtained excellent results.