In Vivo Ultrafast Doppler Imaging Combined with Confocal Microscopy and Behavioral Approaches to Gain Insight into the Central Expression of Peripheral Neuropathy in Trembler-J Mice.

The main human hereditary peripheral neuropathy (Charcot-Marie-Tooth, CMT), manifests in progressive sensory and motor deficits. Mutations in the compact myelin protein gene pmp22 cause more than 50% of all CMTs. CMT1E is a subtype of CMT1 myelinopathy carrying micro-mutations in pmp22. The Trembler-J mice have a spontaneous mutation in pmp22 identical to that present in CMT1E human patients. PMP22 is mainly (but not exclusively) expressed in Schwann cells. Some studies have found the presence of pmp22 together with some anomalies in the CNS of CMT patients. Recently, we identified the presence of higher hippocampal pmp22 expression and elevated levels of anxious behavior in TrJ/+ compared to those observed in wt. In the present paper, we delve deeper into the central expression of the neuropathy modeled in Trembler-J analyzing in vivo the cerebrovascular component by Ultrafast Doppler, exploring the vascular structure by scanning laser confocal microscopy, and analyzing the behavioral profile by anxiety and motor difficulty tests. We have found that TrJ/+ hippocampi have increased blood flow and a higher vessel volume compared with the wild type. Together with this, we found an anxiety-like profile in TrJ/+ and the motor difficulties described earlier. We demonstrate that there are specific cerebrovascular hemodynamics associated with a vascular structure and anxious behavior associated with the TrJ/+ clinical phenotype, a model of the human CMT1E disease.

Quadratic versus linear models to estimate the mean scattering spacing as a function of temperature in ex-vivo tissue.

Previous works have shown the feasibility of temperature estimation during ultrasonic therapy using pulse-echo diagnostic ultrasound. These methods are based on the measurement of thermally induced changes in backscattered RF echoes due to thermal expansion and changes in ultrasonic velocity. They assume a joint contribution of these two parameters and a linear dependence with temperature. In this work, the contributions of velocity changes and thermal expansion to the evolution of the mean scatterer spacing of ex vivo bovine skeletal muscle tissue samples were decoupled. This was achieved by employing an experimental setup which allows measuring the absolute velocity value, using the through-transmission technique in a direct transmission configuration. The mean-scatterer spacing was estimated from spectral analysis of the backscattered signals obtained in pulse-echo mode. We propose a quadratic model of the thermal expansion coefficient to fit the evolution of the mean-scatterer spacing with temperature. The temperature increase estimated by the linear model, in the range of 29.5-47 °C, presents a percentage error (mean square error) of 11 %, while for the quadratic model the error is 4.8 %.

Time reversal of long coda waves: Experiments in the nonlinear regime.

A problem that arises when the time-reversal process is applied in a nonlinear regime is related to the generation of harmonics: conventional piezoelectric transducers cannot work properly at the frequency of the second harmonic. Then, the time-reversed wave cannot be completely emitted. Few works provide a solution to this issue. Thus, we study the alternative of performing a cross correlation of the wavefield. In a linear regime, this procedure is an accurate method for estimating real time-reversal properties. To study both procedures in the nonlinear regime in detail, we measure the wavefield of a wave that (1) traverses a multiple scattering medium, composed by a random set of parallel copper rods and (2) propagates inside a reverberant cavity, consisting of an aluminum case immersed in water. Cross correlation yields a virtually focused wavefield, where the focal width at the frequency of the first, second, and third harmonics can be measured. We compare these values with those obtained in a real time-reversal experiment. Results suggest that both time-reversal procedures are equivalent. In addition, we discuss the possibility of amplitude estimation at the focal spot and the limits of this work based on a theoretical model.

Intensity distribution segmentation in ultrafast Doppler combined with scanning laser confocal microscopy for assessing vascular changes associated with ageing in murine hippocampi.

The hippocampus plays an important role in learning and memory, requiring high-neuronal oxygenation. Understanding the relationship between blood flow and vascular structure-and how it changes with ageing-is physiologically and anatomically relevant. Ultrafast Doppler ([Formula: see text]Doppler) and scanning laser confocal microscopy (SLCM) are powerful imaging modalities that can measure in vivo cerebral blood volume (CBV) and post mortem vascular structure, respectively. Here, we apply both imaging modalities to a cross-sectional and longitudinal study of hippocampi vasculature in wild-type mice brains. We introduce a segmentation of CBV distribution obtained from [Formula: see text]Doppler and show that this mice-independent and mesoscopic measurement is correlated with vessel volume fraction (VVF) distribution obtained from SLCM-e.g., high CBV relates to specific vessel locations with large VVF. Moreover, we find significant changes in CBV distribution and vasculature due to ageing (5 vs. 21 month-old mice), highlighting the sensitivity of our approach. Overall, we are able to associate CBV with vascular structure-and track its longitudinal changes-at the artery-vein, venules, arteriole, and capillary levels. We believe that this combined approach can be a powerful tool for studying other acute (e.g., brain injuries), progressive (e.g., neurodegeneration) or induced pathological changes.

Simplified Green's function for surface waves in quasi-incompressible elastic plates with application to elastography.

Surface wave elastography is a growing method to estimate the elasticity in soft solids. It is particularly useful in the case of agrifoods like meat, cheese, or fruits because it does not require major infrastructure or large equipment and could be developed in portable devices. However, estimating the shear elastic properties from surface wave measurements is not straightforward. The shear wavelength in those materials is cm sized for the excitation frequencies usually employed in elastography (∼102 Hz), and the size of samples is comparable to it. Thus, the surface wave speed is frequency dependent with no direct relation to the shear wave speed. In this work we propose a simplified Green's function for soft solid elastic plates which allows to retrieve the shear elasticity from near field measurements. The model is compared with experimental results obtained in agar-gelatin phantoms and food samples (cheese and bovine liver). The results show a good overall agreement although improvements can be achieved by incorporating diffraction and viscosity to the model.

Vortex dynamics and transport phenomena in stenotic aortic models using Echo-PIV.

Atherosclerosis is the most fatal cardiovascular disease. As disease progresses, stenoses grow inside the arteries blocking their lumen and altering blood flow. Analysing flow dynamics can provide a deeper insight on the stenosis evolution. In this work we combined Eulerian and Lagrangian descriptors to analyze blood flow dynamics and fluid transport in stenotic aortic models with morphology, mechanical and optical properties close to those of real arteries. To this end, vorticity, particle residence time (PRT), particle's final position (FP) and finite time Lyapunov's exponents (FTLE) were computed from the experimental fluid velocity fields acquired using ultrasonic particle imaging velocimetry (Echo-PIV). For the experiments, CT-images were used to create morphological realistic models of the descending aorta with 0%, 35% and 50% occlusion degree with same mechanical properties as real arteries. Each model was connected to a circuit with a pulsatile programmable pump which mimics physiological flow and pressure conditions. The pulsatile frequency was set to ≈0.9 Hz (55 bpm) and the upstream peak Reynolds number (Re) was changed from 1100 to 2000. Flow in the post-stenotic region was composed of two main structures: a high velocity jet over the stenosis throat and a recirculation region behind the stenosis where vortex form and shed. We characterized vortex kinematics showing that vortex propagation velocity increases withRe. Moreover, from the FTLE field we identified Lagrangian coherent structures (i.e. material barriers) that dictate transport behind the stenosis. The size and strength of those barriers increased withReand the occlusion degree. Finally, from the PRT and FP maps, we showed that independently ofRe, the same amount of fluid remains on the stenosis over more than a pulsatile period.

Nonlinear wave propagation through multiple scattering media and virtual time reversal focusing.

In this work, the propagation of an ultrasonic nonlinear wave through a multiple scattering medium is experimentally studied. The interaction between multiple scattering and nonlinear phenomena is analyzed by the cross correlation of the scattered field. This approach corresponds to a virtual time reversal. The cross correlated field is focused in both time and space. In linear regime, it is known that the focal width decreases as the thickness of the multiple scattering medium is increased. In this work, it is shown that this behavior is followed by a nonlinear wave and its harmonics. Moreover, due to the spectral richness of the nonlinear wave, the focal width is reduced in the nonlinear regime. This fact allows for the conclusion that the harmonics propagate following a linear scattering equation, although a nonlinear regime is required to generate them. Beside the experimental work, an estimation on the order of magnitude of the parameters that quantify nonlinearity and scattering phenomena is performed. The estimation shows that the Lighthill-Westervelt equation is as an accurate theoretical model for describing the multiple scattering of a nonlinear wave in the experiments.

Durability study of a gellan gum-based tissue-mimicking phantom for ultrasonic thermal therapy.

Stability and duration of ultrasonic phantoms are still subjects of research. This work presents a tissue-mimicking material (TMM) to evaluate high-intensity therapeutic ultrasound (HITU) devices, composed of gellan gum (matrix), microparticles (scatterers), and chemicals. The ultrasonic velocity and attenuation coefficient were characterized as a function of temperature (range 20 °C-85 °C). The nonlinear parameter B/A was determined by the finite amplitude insertion substitution (FAIS) method, and the shear modulus was determined by a transient elastography technique. The thermal conductivity and specific heat were determined by the line source method. The attenuation was stable for 60 days, and in an almost linear frequency dependence (0.51f0.96 dB cm-1), at 20 °C (1-10 MHz). All other evaluated physical parameters are also close to typical soft tissue values. Longitudinal ultrasonic velocities were between 1.49 and 1.75 mm µs-1, the B/A parameter was 7.8 at 30 °C, and Young's modulus was 23.4 kPa. The thermal conductivity and specific heat values were 0.7 W(m K)-1 and 4.7 kJ(kg K)-1, respectively. Consistent temperature increases and thermal doses occurred under identical HITU exposures. Low cost, longevity, thermal stability, and thermal repeatability make TMM an excellent material for ultrasonic thermal applications. The TMM developed has the potential to assess the efficacy of hyperthermia devices and could be used to adjust the ultrasonic emission of HITU devices.

Development and evaluation of anisotropic and nonlinear aortic models made from clinical images for in vitro experimentation.

In this work we developed a methodology to manufacture a new type of arterial model that could be used in experimental setting instead of excised arteries while developing new imaging modalities. CT-images of the descending aorta were used to create molds with patient specific morphology. A polyvinyl alcohol (PVA) solution with a reinforcing cotton mesh was used to generate the models. The mesh is circumferentially elastic while non-compliant longitudinally and is responsible for the non-linear anisotropic mechanical behavior of the models. Two models were fabricated following the same manufacturing procedure. Their circumferential and longitudinal mechanical properties were evaluated and compared to those of excised healthy pig aortas via tensile testing. A very good agreement was found for the circumferential direction, while the longitudinal direction showed to have a more marked anisotropic behavior compared to the excise arteries. An increase from 113 kPa at 2.5% strain, to 914 kPa at 40% strain was obtained for the models, while the arteries showed an increase from 172 kPa at 2.5% strain to 922 kPa at 38% strain. Furthermore, by plugging the models into a cardiovascular simulator their mechanical response in a more realistic setting was evaluated under static and dynamic pressure conditions by using shear wave elastography (SWE). Static and dynamic experiments showed an increase in the shear modulus as a function of pressure from 61 kPa to 263 kPa, between 20 mmHg and 150 mmHg for Model 1 (similar values within 10% were obtained for Model 2). These values are in good agreement with those reported in the literature for healthy human arteries. To our knowledge the models presented in this study are the first morphologically realistic phantoms that have demonstrated nonlinear and anisotropic elastic behaviors close to those of healthy arteries.

Ex vivo determined experimental correction factor for the ultrasonic source term in the bioheat equation.

The objective of this work is to propose an effective absorption coefficient (αeffec) as an empirical correction factor in the source term of the bioheat equation. The temperature rise in biological tissue due to ultrasound insonification is produced by energy absorption. Usually, the ultrasonic absorption coefficient (αA) is used as a source term in the bioheat equation to quantify the temperature rise, and the effect of scattering is disregarded. The coefficient αeffec includes the scattering contribution as an additional absorption term and should allow us to make a better estimation of the thermal dose (TD), which is important for clinical applications. We simulated the bioheat equation with the source term considering αA or αeffec, and with heating provided by therapeutic ultrasound (1MHz, 2.0Wcm-2) for about 5.5min (temperature range 36-46°C). Experimental data were obtained in similar heating conditions for a bovine muscle tissue (ex vivo) and temperature curves were measured for depths 7, 30, 35, 40 and 45mm. The TD values from the experimental temperature curves at each depth were compared with the numerical solution of the bioheat equation with the classical and corrected source terms. The highest percentual difference between simulated and experimental TD was 42.5% when assuming the classical αA, and 8.7% for the corrected αeffec. The results show that the effective absorption coefficient is a feasible parameter to improve the classical bioheat transfer model, especially for depths larger than the mean free propagation path.

Analysis of the transient surface wave propagation in soft-solid elastic plates.

In dynamic elastography, the goal is to estimate the Young's modulus from audio-frequency wave propagation in soft-tissues. Within this frequency range, the shear wavelength is centimeter-sized while the compressional wavelength is meter-sized. Thus, the experimental data are usually collected in the near-field of the source. Near-field effects have been widely studied for bulk wave propagation. However, the near- and transient-fields of surface and guided waves have received less attention. In this work, the transient surface displacement field in soft-solid elastic plates in vacuum is analyzed. Due to the high Poisson's ratio, mode conversion has special characteristics in soft-solids. They are analyzed through this work where it is shown that the transient-field over the surface can be interpreted by tracing a few reflections. The authors show the existence of a critical distance needed for the formation of Rayleigh-Lamb modes. Below this distance, only direct surface waves propagate without contribution from reflected waves. Thus, the dispersion curve differs from that predicted by Rayleigh-Lamb modes. Instead, the authors propose a model based on the interference of surface waves, which agree with the experimental data. In addition, the conditions needed in order to retrieve the shear wave phase velocity from the surface field are given.

A diffraction correction for storage and loss moduli imaging using radiation force based elastography.

Noninvasive evaluation of the rheological behavior of soft tissues may provide an important diagnosis tool. Nowadays, available commercial ultrasound systems only provide shear elasticity estimation by shear wave speed assessment under the hypothesis of a purely elastic model. However, to fully characterize the rheological behavior of tissues, given by its storage (G') and loss (G″) moduli, it is necessary to estimate both: shear wave speed and shear wave attenuation. Most elastography techniques use the acoustic radiation force to generate shear waves. For this type of source the shear waves are not plane and a diffraction correction is needed to properly estimate the shear wave attenuation. The use of a cylindrical wave approximation to evaluate diffraction has been proposed by other authors before. Here the validity of such approximation is numerically and experimentally revisited. Then, it is used to generate images of G' and G″ in heterogeneous viscoelastic mediums. A simulation algorithm based on the anisotropic and viscoelastic Green's function was used to establish the validity of the cylindrical approximation. Moreover, two experiments were carried out: a transient elastography experiment where plane shear waves were generated using a vibrating plate and a SSI experiment that uses the acoustic radiation force to generate shear waves. For both experiments the shear wave propagation was followed with an ultrafast ultrasound scanner. Then, the shear wave velocity and shear wave attenuation were recovered from the phase and amplitude decay versus distance respectively. In the SSI experiment the cylindrical approximation was applied to correct attenuation due to diffraction effects. The numerical and experimental results validate the use of a cylindrical correction to assess shear wave attenuation. Finally, by applying the cylindrical correction G' and G″ images were generated in heterogeneous phantoms and a preliminary in vivo feasibility study was carried out in the human liver.

Influence of ultrasonic scattering in the calculation of thermal dose in ex-vivo bovine muscular tissues.

This study explores the effect of ultrasound scattering on the temperature increase in phantoms and in samples of ex-vivo biological tissue through the calculation of the thermal dose (TD). Phantoms with different weight percentages of graphite powder (0-1%w/w, different scattering mean free paths, ℓS) and ex-vivo bovine muscle tissue were isonified by therapeutic ultrasound (1 MHz). The TD values were calculated from the first 4 min of experimental temperature curves obtained at several depths and were compared with those acquired from the numerical solution of the bio-heat transfer equation (simulated with 1 MHz and 0.5-2.0 W cm(-2)). The temperature curves suggested that scattering had an important role because the temperature increments were found to be higher for higher percentages of graphite powder (lower ℓS). For example, at a 30-mm depth and a 4-min therapeutic ultrasound application (0.5 W cm(-2)), the TDs (in equivalent minutes at 43 °C) were 7.2, 17.8, and 58.3 for the phantom with ℓS of 4.35, 3.85, and 3.03 mm, respectively. In tissue, the inclusion of only absorption or full attenuation in the bio-heat transfer equation (BHTE) heat source term of the simulation leads to under- or overestimation of the TD, respectively, as compared to the TD calculated from experimental data. The experiments with phantoms (with different scatterer concentrations) and ex-vivo samples show that the high values of TD were caused by the increase of energy absorption due to the lengthening of the propagation path caused by the changing in the propagation regime.

Quantitative shear elasticity imaging from a complex elastic wavefield in soft solids with application to passive elastography.

In passive elastography, the complex physiological noise present in the human body is used to conduct an elastography experiment. In the present work, quantitative shear elasticity imaging from a complex elastic wavefield is demonstrated in soft solids. By correlating the elastic field at different positions, which can be interpreted as a time-reversal experiment, shear waves are virtually focused on any point of the imaging plane. According to the Rayleigh criterion, the focus size is directly related to the shear wave speed and thus to the shear elasticity. To locally retrieve a shear wave speed estimation, analytical and empirical expressions that relate the focus size with the shear wave speed and the frequency band used in the correlation computation are derived. The validity of such expressions is demonstrated numerically and experimentally on a tissue-mimicking phantom consisting of two different elastic layers. The obtained results were in complete agreement with a prior shear wave speed estimation demonstrating the potential of the technique to quantitative shear elasticity assessment using a complex elastic wavefield. Finally, an ultraslow experiment at an imaging rate of 10 Hz shows the technique to be compatible with slow imaging devices such as standard echographs or MRI scanners.

A new high-resolution spectral approach to noninvasively evaluate wall deformations in arteries.

By locally measuring changes on arterial wall thickness as a function of pressure, the related Young modulus can be evaluated. This physical magnitude has shown to be an important predictive factor for cardiovascular diseases. For evaluating those changes, imaging segmentation or time correlations of ultrasonic echoes, coming from wall interfaces, are usually employed. In this paper, an alternative low-cost technique is proposed to locally evaluate variations on arterial walls, which are dynamically measured with an improved high-resolution calculation of power spectral densities in echo-traces of the wall interfaces, by using a parametric autoregressive processing. Certain wall deformations are finely detected by evaluating the echoes overtones peaks with power spectral estimations that implement Burg and Yule Walker algorithms. Results of this spectral approach are compared with a classical cross-correlation operator, in a tube phantom and "in vitro" carotid tissue. A circulating loop, mimicking heart periods and blood pressure changes, is employed to dynamically inspect each sample with a broadband ultrasonic probe, acquiring multiple A-Scans which are windowed to isolate echo-traces packets coming from distinct walls. Then the new technique and cross-correlation operator are applied to evaluate changing parietal deformations from the detection of displacements registered on the wall faces under periodic regime.

Similarities of arterial collagen pressure-diameter relationship in ovine femoral arteries and PLLA vascular grafts.

INTRODUCTION: In-vivo implanted vascular grafts fail due to the mechanical mismatch between the native vessel and the implant. The biomechanical characterization of native vessels provides valuable information towards the development of synthetic grafts. MATERIALS AND METHODS: Five samples of electrospun nanofibrous poly(L-lactic acid)(PLLA) tubular structures were subjected to physiological pulsating pressure using an experimental setup. Four ovine femoral arteries were also tested in the experimental setup under the same conditions. Instantaneous diameter and pressure signals were obtained using gold standard techniques, in order to estimate the dynamic pressure-strain elastic modulus (E(Pε)) of both native vessels and grafts. RESULTS: Synthetic grafts showed a significant increase of E(Pε) (10.57±0.97 to 17.63±2.61 10(6) dyn/cm(2)) when pressure was increased from a range of 50-90 mmHg (elastin-response range) to a range of 100-130 mmHg (collagen-response range). Furthermore, femoral arteries also exhibited a significant increase of EPε (1.66±0.30 to 15.76±4.78 10(6) dyn/cm(2)) with the same pressure variation, showing that both native vessels and synthetic grafts have a similar behavior in the collagen-acting range. CONCLUSION: The mechanical behavior of PLLA vascular grafts was characterized In vitro. However, the procedure can be easily extrapolated to In vivo experiences in conscious and chronically instrumented animals.

Estimation of PSD shifts for high-resolution metrology of thickness micro-changes with possible applications in vessel walls and biological membrane characterization.

Achieving accurate measurements of inflammation levels in tissues or thickness changes in biological membranes (e.g., amniotic sac, parietal pleura) and thin biological walls (e.g., blood vessels) from outside the human body, is a promising research line in the medical area. It would provide a technical basis to study the options for early diagnosis of some serious diseases such as hypertension, atherosclerosis or tuberculosis. Nevertheless, achieving the aim of non-invasive measurement of those scarcely-accessible parameters on patient internal tissues, currently presents many difficulties. The use of high-frequency ultrasonic transducer systems appears to offer a possible solution. Previous studies using conventional ultrasonic imaging have shown this, but the spatial resolution was not sufficient so as to permit a thickness evaluation with clinical significance, which requires an accuracy of a few microns. In this paper a broadband ultrasonic technique, that was recently developed by the authors to address other non-invasive medical detection problems (by integrating a piezoelectric transducer into a spectral measuring system), is extended to our new objective; the aim is its application to the thickness measurement of sub-millimeter membranes or layers made of materials similar to some biological tissues (phantoms). The modeling and design rules of such a transducer system are described, and various methods of estimating overtones location in the power spectral density (PSD) are quantitatively assessed with transducer signals acquired using piezoelectric systems and also generated from a multi-echo model. Their effects on the potential resolution of the proposed thickness measuring tool, and their capability to provide accuracies around the micron are studied in detail. Comparisons are made with typical tools for extracting spatial parameters in laminar samples from echo-waveforms acquired with ultrasonic transducers. Results of this advanced measurement spectral tool are found to improve the performance of typical cross-correlation methods and provide reliable and high-resolution estimations.

Application of 1-D transient elastography for the shear modulus assessment of thin-layered soft tissue: comparison with supersonic shear imaging technique.

Elasticity estimation of thin-layered soft tissues has gained increasing interest propelled by medical applications like skin, corneal, or arterial wall shear modulus assessment. In this work, the authors propose one-dimensional transient elastography (1DTE) for the shear modulus assessment of thin-layered soft tissue. Experiments on three phantoms with different elasticities and plate thicknesses were performed. First, using 1DTE, the shear wave speed dispersion curve inside the plate was obtained and validated with finite difference simulation. No dispersive effects were observed and the shear wave speed was directly retrieved from time-of-flight measurements. Second, the supersonic shear imaging (SSI) technique (considered to be a gold standard) was performed. For the SSI technique, the propagating wave inside the plate is guided as a Lamb wave. Experimental SSI dispersion curves were compared with finite difference simulation and fitted using a generalized Lamb model to retrieve the plate bulk shear wave speed. Although they are based on totally different mechanical sources and induce completely different diffraction patterns for the shear wave propagation, the 1DTE and SSI techniques resulted in similar shear wave speed estimations. The main advantage of the 1DTE technique is that bulk shear wave speed can be directly retrieved without requiring a dispersion model.

Myelinating and demyelinating phenotype of Trembler-J mouse (a model of Charcot-Marie-Tooth human disease) analyzed by atomic force microscopy and confocal microscopy.

The accumulation of misfolded proteins is associated with various neurodegenerative conditions. Mutations in PMP-22 are associated with the human peripheral neuropathy, Charcot-Marie-Tooth Type 1A (CMT1A). PMP-22 is a short-lived 22 kDa glycoprotein, which plays a key role in the maintenance of myelin structure and compaction, highly expressed by Schwann cells. It forms aggregates when the proteasome is inhibited or the protein is mutated. This study reports the application of atomic force microscopy (AFM) as a detector of profound topographical and mechanical changes in Trembler-J mouse (CMT1A animal model). AFM images showed topographical differences in the extracellular matrix and basal lamina organization of Tr-J/+ nerve fibers. The immunocytochemical analysis indicated that PMP-22 protein is associated with type IV collagen (a basal lamina ubiquitous component) in the Tr-J/+ Schwann cell perinuclear region. Changes in mechanical properties of single myelinating Tr-J/+ nerve fibers were investigated, and alterations in cellular stiffness were found. These results might be associated with F-actin cytoskeleton organization in Tr-J/+ nerve fibers. AFM nanoscale imaging focused on topography and mechanical properties of peripheral nerve fibers might provide new insights into the study of peripheral nervous system diseases.