Inducible nonhuman primate models of retinal degeneration for testing end-stage therapies.

The anatomical differences between the retinas of humans and most animal models pose a challenge for testing novel therapies. Nonhuman primate (NHP) retina is anatomically closest to the human retina. However, there is a lack of relevant NHP models of retinal degeneration (RD) suitable for preclinical studies. To address this unmet need, we generated three distinct inducible cynomolgus macaque models of RD. We developed two genetically targeted strategies using optogenetics and CRISPR-Cas9 to ablate rods and mimic rod-cone dystrophy. In addition, we created an acute model by physical separation of the photoreceptors and retinal pigment epithelium using a polymer patch. Among the three models, the CRISPR-Cas9-based approach was the most advantageous model in view of recapitulating disease-specific features and its ease of implementation. The acute model, however, resulted in the fastest degeneration, making it the most relevant model for testing end-stage vision restoration therapies such as stem cell transplantation.

Electrical and Optical Characterization of SAW Sensors Coated with Parylene C and Their Analysis Using the Coupling-of-Modes (COM) Theory.

In this paper, we present how complementary characterization techniques, such as electrical measurements with a vector network analyzer (VNA), optical measurements with a laser Doppler vibrometer (LDV), and numerical simulations with the finite element method, coupled with spectral domain analysis (FEMSDA), allow us to independently access different properties of a SAW device and fully characterize its operation using the coupling-of-modes theory (COM). A set of chemical SAW sensors coated with parylene C layers of different thicknesses (1, 1.5, and 2 µm) and an uncoated sensor were used as test samples. The sensors represent dual-channel electroacoustic delay lines operating in the vicinity of 77 MHz. The IDTs consist of split aluminum electrodes deposited on a AT-cut quartz substrate. The thickness-dependent influence of the parylene C layer was observed on the operating frequency (SAW velocity), static capacitance, attenuation, crosstalk, and reflection coefficient. COM parameters were reported for the four cases considered; measured and simulated data show good agreement. The presented approach is suitable for the design, characterization, and validation of polymer film-coated SAW sensors.

Neural tissue-microelectrode interaction: Brain micromotion, electrical impedance, and flexible microelectrode insertion.

Insertion of a microelectrode into the brain to record/stimulate neurons damages neural tissue and blood vessels and initiates the brain's wound healing response. Due to the large difference between the stiffness of neural tissue and microelectrode, brain micromotion also leads to neural tissue damage and associated local immune response. Over time, following implantation, the brain's response to the tissue damage can result in microelectrode failure. Reducing the microelectrode's cross-sectional dimensions to single-digit microns or using soft materials with elastic modulus close to that of the neural tissue are effective methods to alleviate the neural tissue damage and enhance microelectrode longevity. However, the increase in electrical impedance of the microelectrode caused by reducing the microelectrode contact site's dimensions can decrease the signal-to-noise ratio. Most importantly, the reduced dimensions also lead to a reduction in the critical buckling force, which increases the microelectrode's propensity to buckling during insertion. After discussing brain micromotion, the main source of neural tissue damage, surface modification of the microelectrode contact site is reviewed as a key method for addressing the increase in electrical impedance issue. The review then focuses on recent approaches to aiding insertion of flexible microelectrodes into the brain, including bending stiffness modification, effective length reduction, and application of a magnetic field to pull the electrode. An understanding of the advantages and drawbacks of the developed strategies offers a guide for dealing with the buckling phenomenon during implantation.

A detailed in-vitro study of Retinal pigment epithelium's growth as seen from the perspective of impedance spectroscopy analysis.

In this paper, we present a method to assess growth and maturation phases of the Retinal Pigment Epithelium (RPE) in-vitro at the cell layer level using impedance spectroscopy measurements on platinum electrodes. We extracted relevant parameters from an electrical circuit model fitted with the measured spectra. Based on microscopic imaging, the growth state of an independent culture developing in the same conditions is used as reference. We show that the confluence point is identified from a graphical analysis of the spectra transition as well as by observing a reconstructed parameter representing the average capacitance of the cell layer. More generally, this work presents a detailed investigation on how cell culture's state relates with either model parameter analysis or with graphical analysis of the measured spectra over a wide frequency band. While applied to the RPE, this work is also suitable for the study of any kind of monolayer epithelial cells growth.

Impedance spectroscopy study of the retinal pigment epithelium: Application to the monitoring of blue light exposure effect on A2E-loaded in-vitro cell cultures.

In age-related macular degeneration, the retinal pigment epithelium can be damaged by light acting on photosensitizers like N-retinylidene-N-retinylethanolamine (A2E). In this paper, the underlying cellular mechanism of lesion at the cell layer scale is analyzed by impedance spectroscopy. Retinal pigment epithelium (RPE) cells are cultured on top of custom-made electrodes capable of taking impedance measurements, with the help of a custom-made electronic setup but without the use of any chemical markers. An incubator is used to house the cells growing on the electrodes. An electrical model circuit is presented and linked to the constituents of the cell layer in which various electrical elements have been defined including a constant phase element (CPE) associated to the interface between the cell layer and the electrolyte. Their values are extracted from the fitted model of the measured impedance spectra. In this paper, we first investigate which parameters of the model can be analyzed independently. In that way, the parameter's evolution is examined with respect to two different targeted changes of the epithelium: 1. degradation of tight junctions between cells by extracellular calcium sequestration with Ethylenediaminetetraacetic acid (EDTA); 2. application of high amplitude short length electric field pulses. Based on the results obtained showing a clear relation between the model and the physiological state of the cell layer, the same procedure is applied to blue light exposure experiment. When A2E-loaded cells are exposed to blue light, the model parameters indicate, as expected, a clear degradation of the cell layer opposed to a relative stability of the not loaded ones.

Protuberant Electrode Structures for Subretinal Electrical Stimulation: Modeling, Fabrication and in vivo Evaluation.

Many neural interfaces used for therapeutic applications are based on extracellular electrical stimulation to control cell polarization and thus functional activity. Amongst them, retinal implants have been designed to restore visual perception in blind patients affected by photoreceptor degeneration diseases, such as age-related macular degeneration (AMD) or retinitis pigmentosa (RP). While designing such a neural interface, several aspects must be taken into account, like the stimulation efficiency related to the current distribution within the tissue, the bio-interface optimization to improve resolution and tissue integration, and the material biocompatibility associated with long-term aging. In this study, we investigate the use of original microelectrode geometries for subretinal stimulation. The proposed structures combine the use of 3D wells with protuberant mushroom shaped electrode structures in the bottom, implemented on a flexible substrate that allows the in vivo implantation of the devices. These 3D microelectrode structures were first modeled using finite element analysis. Then, a specific microfabrication process compatible with flexible implants was developed to create the 3D microelectrode structures. These structures were tested in vivo to check the adaptation of the retinal tissue to them. Finally, preliminary in vivo stimulation experiments were performed.

Development of Diamond and Silicon MEMS Sensor Arrays with Integrated Readout for Vapor Detection.

This paper reports on the development of an autonomous instrument based on an array of eight resonant microcantilevers for vapor detection. The fabricated sensors are label-free devices, allowing chemical and biological functionalization. In this work, sensors based on an array of silicon and synthetic diamond microcantilevers are sensitized with polymeric films for the detection of analytes. The main advantage of the proposed system is that sensors can be easily changed for another application or for cleaning since the developed gas cell presents removable electrical connections. We report the successful application of our electronic nose approach to detect 12 volatile organic compounds. Moreover, the response pattern of the cantilever arrays is interpreted via principal component analysis (PCA) techniques in order to identify samples.

Monitoring the evolution of boron doped porous diamond electrode on flexible retinal implant by OCT and in vivo impedance spectroscopy.

Nanocrystalline Boron doped Diamond proved to be a very attractive material for neural interfacing, especially with the retina, where reduce glia growth is observed with respect to other materials, thus facilitating neuro-stimulation over long terms. In the present study, we integrated diamond microelectrodes on a polyimide substrate and investigated their performances for the development of neural prosthesis. A full description of the microfabrication of the implants is provided and their functionalities are assessed using cyclic voltammetry and electrochemical impedance spectroscopy. A porous structure of the electrode surface was thus revealed and showed promising properties for neural recording or stimulation. Using the flexible implant, we showed that is possible to follow in vivo the evolution of the electric contact between the diamond electrodes and the retina over 4months by using electrochemical impedance spectroscopy. The position of the implant was also monitored by optical coherence tomography to corroborate the information given by the impedance measurements. The results suggest that diamond microelectrodes are very good candidates for retinal prosthesis.

Synthetic 3D diamond-based electrodes for flexible retinal neuroprostheses: Model, production and in vivo biocompatibility.

Two retinal implants have recently received the CE mark and one has obtained FDA approval for the restoration of useful vision in blind patients. Since the spatial resolution of current vision prostheses is not sufficient for most patients to detect faces or perform activities of daily living, more electrodes with less crosstalk are needed to transfer complex images to the retina. In this study, we modelled planar and three-dimensional (3D) implants with a distant ground or a ground grid, to demonstrate greater spatial resolution with 3D structures. Using such flexible 3D implant prototypes, we showed that the degenerated retina could mould itself to the inside of the wells, thereby isolating bipolar neurons for specific, independent stimulation. To investigate the in vivo biocompatibility of diamond as an electrode or an isolating material, we developed a procedure for depositing diamond onto flexible 3D retinal implants. Taking polyimide 3D implants as a reference, we compared the number of neurones integrating the 3D diamond structures and their ratio to the numbers of all cells, including glial cells. Bipolar neurones were increased whereas there was no increase even a decrease in the total cell number. SEM examinations of implants confirmed the stability of the diamond after its implantation in vivo. This study further demonstrates the potential of 3D designs for increasing the resolution of retinal implants and validates the safety of diamond materials for retinal implants and neuroprostheses in general.

3D-nanostructured boron-doped diamond for microelectrode array neural interfacing.

The electrode material is a key element in the design of long-term neural implants and neuroprostheses. To date, the ideal electrode material offering high longevity, biocompatibility, low-noise recording and high stimulation capabilities remains to be found. We show that 3D-nanostructured boron doped diamond (BDD), an innovative material consisting in a chemically stable material with a high aspect ratio structure obtained by encapsulation of a carbon nanotube template within two BDD nanolayers, allows neural cell attachment, survival and neurite extension. Further, we developed arrays of 20-µm-diameter 3D-nanostructured BDD microelectrodes for neural interfacing. These microelectrodes exhibited low impedances and low intrinsic recording noise levels. In particular, they allowed the detection of low amplitude (10-20 µV) local-field potentials, single units and multiunit bursts neural activity in both acute whole embryonic hindbrain-spinal cord preparations and long-term hippocampal cell cultures. Also, cyclic voltammetry measurements showed a wide potential window of about 3 V and a charge storage capacity of 10 mC.cm(-2), showing high potentiality of this material for neural stimulation. These results demonstrate the attractiveness of 3D-nanostructured BDD as a novel material for neural interfacing, with potential applications for the design of biocompatible neural implants for the exploration and rehabilitation of the nervous system.

[Restoring vision in blind patients following photoreceptor degeneration: clinical results and future challenges]. / Restaurer la vision de patients aveugles avec des prothèses rétiniennes : Résultats cliniques et défis futurs.

Retinal prostheses aim at restoring vision in patients blind from photoreceptor degeneration by electrically stimulating the residual retinal tissue. Currently, the most efficient implants are either inserted in the subretinal space or on the vitreal side of the retina (epi-retinal). Although the residual tissue can partly degenerate, it was shown that acute stimulation of residual neurones can induce visual percepts. Recently, a clinical trial with the epiretinal Argus2 device (60 electrodes) from the company 2nd Sight enabled most patients to orient and find light targets, some even reading words. This device has received a CE mark. Surprisingly, when the subretinal implant from the company Retina Implant AG displaying many more electrodes (1500 electrodes) was evaluated in clinical trials, the patient visual performances were fairly similar. The restored visual performances of the patients demonstrate that blind patients can recover some visual function when their residual retina is properly stimulated. However, the resolution is not yet sufficient to perform complex tasks such as autonomous locomotion, face identification or text reading. Several challenges remain to generate an increase in pixel density corresponding to the increase in electrode number and density. These challenges include the stimulation modality, the tissue/implant interface design, the electrode materials, and the visual information encoder. This review will discuss these great challenges after introducing the major clinical results.

Multichannel boron doped nanocrystalline diamond ultramicroelectrode arrays: design, fabrication and characterization.

We report on the fabrication and characterization of an 8 × 8 multichannel Boron Doped Diamond (BDD) ultramicro-electrode array (UMEA). The device combines both the assets of microelectrodes, resulting from conditions in mass transport from the bulk solution toward the electrode, and of BDD's remarkable intrinsic electrochemical properties. The UMEAs were fabricated using an original approach relying on the selective growth of diamond over pre-processed 4 inches silicon substrates. The prepared UMEAs were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results demonstrated that the electrodes have exhibited a very fast electrode transfer rate (k(0)) up to 0.05 cm·s(-1) (in a fast redox couple) and on average, a steady state limiting current (in a 0.5 M potassium chloride aqueous solution containing 1 mM Fe(CN)(6)(4-) ion at 100 mV·s(-1)) of 1.8 nA. The UMEAs are targeted for electrophysiological as well as analytical applications.

Combined macro-/mesoporous microelectrode arrays for low-noise extracellular recording of neural networks.

Microelectrode arrays (MEAs) are appealing tools to probe large neural ensembles and build neural prostheses. Microelectronics microfabrication technologies now allow building high-density MEAs containing several hundreds of microelectrodes. However, several major problems become limiting factors when the size of the microelectrodes decreases. In particular, regarding recording of neural activity, the intrinsic noise level of a microelectrode dramatically increases when the size becomes small (typically below 20-µm diameter). Here, we propose to overcome this limitation using a template-based, single-scale meso- or two-scale macro-/mesoporous modification of the microelectrodes, combining the advantages of an overall small geometric surface and an active surface increased by several orders of magnitude. For this purpose, standard platinum MEAs were covered with a highly porous platinum overlayer obtained by lyotropic liquid crystal templating possibly in combination with a microsphere templating approach. These porous coatings were mechanically more robust than Pt-black coating and avoid potential toxicity issues. They had a highly increased active surface, resulting in a noise level ∼3 times smaller than that of conventional flat electrodes. This approach can thus be used to build highly dense arrays of small-size microelectrodes for sensitive neural signal detection.

Focalizing electrical neural stimulation with penetrating microelectrode arrays: a modeling study.

Penetrating neural probes are considered for neuroprosthetic devices to restore sensory or motor functions of the CNS using electrical neural microstimulation. These multielectrode systems require optimal electrode configurations to allow precise and focused tissue activation. Combining a finite element model of the spinal cord and compartmentalized models of both simple and complex neuron morphologies, we evaluated the use of the "ground surface" configuration, which consists in the integration of a conductive layer on the front side of electrode shanks, for the return of the stimulation current. Compared to the classical monopolar and bipolar configurations, this strategy resulted in a focalization of both the potential field and the threshold-distance curves. The improvement in focalization was highest for lowest impedance of the ground surface. Moreover, the gain in focality was highest on the side of the shank opposite to the electrode, so that only the neurons located in front of stimulation electrode were activated. This focalizing strategy will allow the design of new microstimulation paradigms aiming at precisely targeting the CNS with complex spatio-temporal stimulation patterns, which could benefit to future stimulation-based neuroprosthesis.

High sensitivity of diamond resonant microcantilevers for direct detection in liquids as probed by molecular electrostatic surface interactions.

Resonant microcantilevers have demonstrated that they can play an important role in the detection of chemical and biological agents. Molecular interactions with target species on the mechanical microtransducers surface generally induce a change of the beam's bending stiffness, resulting in a shift of the resonance frequency. In most biochemical sensor applications, cantilevers must operate in liquid, even though damping deteriorates the vibrational performances of the transducers. Here we focus on diamond-based microcantilevers since their transducing properties surpass those of other materials. In fact, among a wide range of remarkable features, diamond possesses exceptional mechanical properties enabling the fabrication of cantilever beams with higher resonant frequencies and Q-factors than when made from other conventional materials. Therefore, they appear as one of the top-ranked materials for designing cantilevers operating in liquid media. In this study, we evaluate the resonator sensitivity performances of our diamond microcantilevers using grafted carboxylated alkyl chains as a tool to investigate the subtle changes of surface stiffness as induced by electrostatic interactions. Here, caproic acid was immobilized on the hydrogen-terminated surface of resonant polycrystalline diamond cantilevers using a novel one-step grafting technique that could be also adapted to several other functionalizations. By varying the pH of the solution one could tune the -COO(-)/-COOH ratio of carboxylic acid moieties immobilized on the surface, thus enabling fine variations of the surface stress. We were able to probe the cantilevers resonance frequency evolution and correlate it with the ratio of -COO(-)/-COOH terminations on the functionalized diamond surface and consequently the evolution of the electrostatic potential over the cantilever surface. The approach successfully enabled one to probe variations in cantilevers bending stiffness from several tens to hundreds of millinewtons/meter, thus opening the way for diamond microcantilevers to direct sensing applications in liquids. The evolution of the diamond surface chemistry was also investigated using X-ray photoelectron spectroscopy.

BioMEA: a versatile high-density 3D microelectrode array system using integrated electronics.

Microelectrode arrays (MEAs) offer a powerful tool to both record activity and deliver electrical microstimulations to neural networks either in vitro or in vivo. Microelectronics microfabrication technologies now allow building high-density MEAs containing several hundreds of microelectrodes. However, dense arrays of 3D micro-needle electrodes, providing closer contact with the neural tissue than planar electrodes, are not achievable using conventional isotropic etching processes. Moreover, increasing the number of electrodes using conventional electronics is difficult to achieve into compact devices addressing all channels independently for simultaneous recording and stimulation. Here, we present a full modular and versatile 256-channel MEA system based on integrated electronics. First, transparent high-density arrays of 3D-shaped microelectrodes were realized by deep reactive ion etching techniques of a silicon substrate reported on glass. This approach allowed achieving high electrode aspect ratios, and different shapes of tip electrodes. Next, we developed a dedicated analog 64-channel Application Specific Integrated Circuit (ASIC) including one amplification stage and one current generator per channel, and analog output multiplexing. A full modular system, called BIOMEA, has been designed, allowing connecting different types of MEAs (64, 128, or 256 electrodes) to different numbers of ASICs for simultaneous recording and/or stimulation on all channels. Finally, this system has been validated experimentally by recording and electrically eliciting low-amplitude spontaneous rhythmic activity (both LFPs and spikes) in the developing mouse CNS. The availability of high-density MEA systems with integrated electronics will offer new possibilities for both in vitro and in vivo studies of large neural networks.