Emilie Macé

VISCOELASTIC AND ANISOTROPIC MECHANICAL PROPERTIES OF IN VIVO MUSCLE TISSUE ASSESSED BY SUPERSONIC SHEAR IMAGING

The in vivo assessment of the biomechanical properties of the skeletal muscle is a complex issue because the muscle is an anisotropic, viscoelastic and dynamic medium. In this article, these mechanical properties are characterized for the brachialis muscle in vivo using a noninvasive ultrasound-based technique. This supersonic shear imaging technique combines an ultra-fast ultrasonic system and the remote generation of transient mechanical forces into tissue via the radiation force of focused ultrasonic beams. Such an ultrasonic radiation force is induced deep within the muscle by a conventional ultrasonic probe and the resulting shear waves are then imaged with the same probe (5 MHz) at an ultra-fast framerate (up to 5000 frames/s). Local tissue velocity maps are obtained with a conventional speckle tracking technique and provide a full movie of the shear wave propagation through the entire muscle. Shear wave group velocities are then estimated using a time of flight algorithm. This approach provides a complete set of quantitative and in vivo parameters describing the muscles mechanical properties as a function of active voluntary contraction as well as passive extension of healthy volunteers. Anisotropic properties are also estimated by tilting the probe head with respects to the main muscular fibers direction. Finally, the dispersion of the shear waves is studied for these different configurations and shear modulus and shear viscosity are quantitatively assessed assuming the viscoelastic Voigts model.

Functional ultrasound imaging of the brain.

We present functional ultrasound (fUS), a method for imaging transient changes in blood volume in the whole brain at better spatiotemporal resolution than with other functional brain imaging modalities. fUS uses plane-wave illumination at high frame rate and can measure blood volumes in smaller vessels than previous ultrasound methods. fUS identifies regions of brain activation and was used to image whisker-evoked cortical and thalamic responses and the propagation of epileptiform seizures in the rat brain.

Functional ultrasound imaging of the brain: theory and basic principles

Hemodynamic changes in the brain are often used as surrogates of neuronal activity to infer the loci of brain activity. A major limitation of conventional Doppler ultrasound for the imaging of these changes is that it is not sensitive enough to detect the blood flow in small vessels where the major part of the hemodynamic response occurs. Here, we present a μDoppler ultrasound method able to detect and map the cerebral blood volume (CBV) over the entire brain with an important increase in sensitivity. This method is based on imaging the brain at an ultrafast frame rate (1 kHz) using compounded plane wave emissions. A theoretical model demonstrates that the gain in sensitivity of the μDoppler method is due to the combination of 1) the high signal-to-noise ratio of the gray scale images, resulting from the synthetic compounding of backscattered echoes; and 2) the extensive signal averaging enabled by the high temporal sampling of ultrafast frame rates. This μDoppler imaging is performed in vivo on trepanned rats without the use of contrast agents. The resulting images reveal detailed maps of the rat brain vascularization with an acquisition time as short as 320 ms per slice. This new method is the basis for a real-time functional ultrasound (fUS) imaging of the brain.

Targeting channelrhodopsin-2 to ON-bipolar cells with vitreally administered AAV Restores ON and OFF visual responses in blind mice.

Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of retinal neurons mediated by adeno-associated virus (AAV) gene therapy has the potential to restore vision regardless of patient-specific mutations. The challenge for clinical translatability is to restore a vision as close to natural vision as possible, while using a surgically safe delivery route for the fragile degenerated retina. To preserve the visual processing of the inner retina, we targeted ON bipolar cells, which are still present at late stages of disease. For safe gene delivery, we used a recently engineered AAV variant that can transduce the bipolar cells after injection into the eyes easily accessible vitreous humor. We show that AAV encoding channelrhodopsin under the ON bipolar cell-specific promoter mediates long-term gene delivery restricted to ON-bipolar cells after intravitreal administration. Channelrhodopsin expression in ON bipolar cells leads to restoration of ON and OFF responses at the retinal and cortical levels. Moreover, light-induced locomotory behavior is restored in treated blind mice. Our results support the clinical relevance of a minimally invasive AAV-mediated optogenetic therapy for visual restoration.

In Vivo Mapping of Brain Elasticity in Small Animals Using Shear Wave Imaging

A combination of radiation force and ultrafast ultra-sound imaging is used to both generate and track the propagation of a shear wave in the brain whose local speed is directly related to stiffness, characterized by the dynamic shear modulus G*. When performed on trepanated rats, this approach called shear wave imaging (SWI) provides 3-D brain elasticity maps reaching a spatial resolution of 0.7 mm × 1 mm × 0.4 mm with a good reproducibility (<;13%). The dynamic shear modulus of brain tissues exhibits values in the 2-25 kPa range with a mean value of 12 kPa and is quantified for different anatomical regions. The anisotropy of the shear wave propagation is studied and the first in vivo anisotropy map of brain elasticity is provided. The propagation is found to be isotropic in three gray matter regions but highly anisotropic in two white matter regions. The good temporal resolution (~10 ms per acquisition) of SWI also allows a dynamic estimation of brain elasticity to within a single cardiac cycle, showing that brain pulsatility does not transiently modify local elasticity. SWI proves its potential for the study of pathological modifications of brain elasticity both in small animal models and in clinical intra-operative imaging.

Imaging of perfusion, angiogenesis, and tissue elasticity after stroke.

Blood flow interruption in a cerebral artery causes brain ischemia and induces dramatic changes of perfusion and metabolism in the corresponding territory. We performed in parallel positron emission tomography (PET) with [15O]H2O, single photon emission computed tomography (SPECT) with [99mTc]hexamethylpropylene-amino-oxime ([99mTc]HMPAO) and ultrasonic ultrafast shear wave imaging (SWI) during, immediately after, and 1, 2, 4, and 7 days after middle cerebral artery occlusion (MCAO) in rats. Positron emission tomography and SPECT showed initial hypoperfusion followed by recovery at immediate reperfusion, hypoperfusion at day 1, and hyperperfusion at days 4 to 7. Hyperperfusion interested the whole brain, including nonischemic areas. Immunohistochemical analysis indicated active angiogenesis at days 2 to 7, strongly suggestive that hyperperfusion was supported by an increase in microvessel density in both brain hemispheres after ischemia. The SWI detected elastic changes of cerebral tissue in the ischemic area as early as day 1 after MCAO appearing as a softening of cerebral tissue whose local internal elasticity decreased continuously from day 1 to 7. Taken together, these results suggest that hyperperfusion after cerebral ischemia is due to formation of neovessels, and indicate that brain softening is an early and continuous process. The SWI is a promising novel imaging method for monitoring the evolution of cerebral ischemia over time in animals.

Red‐shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina

Targeting the photosensitive ion channel channelrhodopsin‐2 (ChR2) to the retinal circuitry downstream of photoreceptors holds promise in treating vision loss caused by retinal degeneration. However, the high intensity of blue light necessary to activate channelrhodopsin‐2 exceeds the safety threshold of retinal illumination because of its strong potential to induce photochemical damage. In contrast, the damage potential of red‐shifted light is vastly lower than that of blue light. Here, we show that a red‐shifted channelrhodopsin (ReaChR), delivered by AAV injections in blind rd1 mice, enables restoration of light responses at the retinal, cortical, and behavioral levels, using orange light at intensities below the safety threshold for the human retina. We further show that postmortem macaque retinae infected with AAV‐ReaChR can respond with spike trains to orange light at safe intensities. Finally, to directly address the question of translatability to human subjects, we demonstrate for the first time, AAV‐ and lentivirus‐mediated optogenetic spike responses in ganglion cells of the postmortem human retina.

Real-time imaging of brain activity in freely moving rats using functional ultrasound

Innovative imaging methods help to investigate the complex relationship between brain activity and behavior in freely moving animals. Functional ultrasound (fUS) is an imaging modality suitable for recording cerebral blood volume (CBV) dynamics in the whole brain but has so far been used only in head-fixed and anesthetized rodents. We designed a fUS device for tethered brain imaging in freely moving rats based on a miniaturized ultrasound probe and a custom-made ultrasound scanner. We monitored CBV changes in rats during various behavioral states such as quiet rest, after whisker or visual stimulations, and in a food-reinforced operant task. We show that fUS imaging in freely moving rats could efficiently decode brain activity in real time.

Multiplexed computations in retinal ganglion cells of a single type

In the early visual system, cells of the same type perform the same computation in different places of the visual field. How these cells code together a complex visual scene is unclear. A common assumption is that cells of a single-type extract a single-stimulus feature to form a feature map, but this has rarely been observed directly. Using large-scale recordings in the rat retina, we show that a homogeneous population of fast OFF ganglion cells simultaneously encodes two radically different features of a visual scene. Cells close to a moving object code quasilinearly for its position, while distant cells remain largely invariant to the object’s position and, instead, respond nonlinearly to changes in the object’s speed. We develop a quantitative model that accounts for this effect and identify a disinhibitory circuit that mediates it. Ganglion cells of a single type thus do not code for one, but two features simultaneously. This richer, flexible neural map might also be present in other sensory systems.Retinal ganglion cell subtypes are traditionally thought to encode a single visual feature across the visual field to form a feature map. Here the authors show that fast OFF ganglion cells in fact respond to two visual features, either object position or speed, depending on the stimulus location.

Ultrafast compound doppler imaging: A new approach of doppler flow analysis

This work applies the concept of compounded plane wave transmissions at very high frames rates of some KHz for ultrafast Doppler analysis over a large region of interest. As this compound imaging method has a similar quality to the standard focusing method but is 10 times faster, it is possible to generate fast Doppler images at frame rates of 300Hz. This frame rate is able to visualize transient phenomena and to display duplex modes with simultaneous color and spectrum analysis for each pixel of the image. The interest of the method is not restricted to high velocity flows; by optimizing the quality of the ultrasonic compounded image, it is possible to image very small velocity flows. This method is applied to a functional imaging of the rat brain by detecting changes in the flow after a drug injection.