James A. Wiler

Surface modification of neural recording electrodes with conducting polymer/biomolecule blends

The interface between micromachined neural microelectrodes and neural tissue plays an important role in chronic in vivo recording. Electrochemical polymerization was used to optimize the surface of the metal electrode sites. Electrically conductive polymers (polypyrrole) combined with biomolecules having cell adhesion functionality were deposited with great precision onto microelectrode sites of neural probes. The biomolecules used were a silk-like polymer having fibronectin fragments (SLPF) and nonapeptide CDPGYIGSR. The existence of protein polymers and peptides in the coatings was confirmed by reflective microfocusing Fourier transform infrared spectroscopy (FTIR). The morphology of the coating was rough and fuzzy, providing a high density of bioactive sites for interaction with neural cells. This high interfacial area also helped to lower the impedance of the electrode site and, consequently, to improve the signal transport. Impedance spectroscopy showed a lowered magnitude and phase of impedance around the biologically relevant frequency of 1 kHz. Cyclic voltammetry demonstrated the intrinsic redox reaction of the doped polypyrrole and the increased charge capacity of the coated electrodes. Rat glial cells and human neuroblastoma cells were seeded and cultured on neural probes with coated and uncoated electrodes. Glial cells appeared to attach better to polypyrrole/SLPF-coated electrodes than to uncoated gold electrodes. Neuroblastoma cells grew preferentially on and around the polypyrrole/CDPGYIGSR-coated electrode sites while the polypyrrole/CH(3)COO(-)-coated sites on the same probe did not show a preferential attraction to the cells. These results indicate that we can adjust the chemical composition, morphology, electronic transport, and bioactivity of polymer coatings on electrode surfaces on a multichannel micromachined neural probe by controlling electrochemical deposition conditions.

In vivo studies of polypyrrole/peptide coated neural probes.

Neural probes are micromachined multichannel electrode arrays that facilitate the functional stimulation and recording of neurons in the peripheral and central nervous system. For long-term implantations, surface modification is necessary for maintaining the stable connection between electrodes and neurons. The conductive polymer polypyrrole (PPy) and synthetic peptide DCDPGYIGSR were co-deposited on the electrode surface by electrochemical polymerization. The stability of PPy/DCDPGYIGSR coatings was tested in soaking experiments. It was found that the peptide was entrapped in the PPy film and did not diffuse away within 7 weeks of soaking in DI water. Coated probes were implanted in guinea pig brain for periods of 1, 2 and 3 weeks. Recording tests were performed and the impedance was monitored. The explanted probes and tissue were examined by immunocytochemical studies. Significantly more neurofilament positive staining was found on the coated electrode which indicated that the coatings had established strong connections with the neuronal structure in vivo. Good recordings were obtained from the coated sites that had neurons attached. First week tissue sections had no significant gliosis. In week 2, a layer of non-neuronal tissue consisting of mostly meningeal fibroblasts and ECM protein including at least fibronectin was formed around the probe tracks of both coated and uncoated probes. Astrocytes started to form a loosely organized layer by the end of the third week.

Conducting polymers on hydrogel-coated neural electrode provide sensitive neural recordings in auditory cortex

Recently, a significant amount of effort has been dedicated to understanding factors that influence the functionality of bio-electronic sensors and to development of novel coating technologies for modifying biosensor surfaces. Due to its well-known biocompatibility, alginate hydrogel (HG) has been used as a coating material on neural electrodes for promoting intimate cellular integration, providing a scaffold for local drug delivery, and creating a mechanical buffer between hard electrodes and the soft tissues of the central nervous system. However, neural signal recordings using HG-coated electrodes in animal models are still poorly evaluated. Here, we investigated the effect of the proximity of source neurons around the electrode sites using HG coatings with various thicknesses deposited on microfabricated electrodes, implanted in auditory cortex of guinea pigs. We also evaluated the role of the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) in improving the recording functionality of the HG-coated neural electrodes. A significant loss in recording functionality was observed with thicker HG coatings, as determined by the number of clearly detectable units (30% with 80 microm thick coatings) and average signal-to-noise ratios (3.91 with 80 microm thick coatings). However, deposition of the conducting polymer PEDOT on the electrode sites restored the lost functionality of the electrodes caused by the HG coatings (30 microm). These conducting polymer/HG coatings have the potential to improve long-term performance of the neural electrodes not only by improving the electrode biocompatibility but also by facilitating more efficient signal transmission.

Transgenic BDNF induces nerve fiber regrowth into the auditory epithelium in deaf cochleae.

Sensory organs typically use receptor cells and afferent neurons to transduce environmental signals and transmit them to the CNS. When sensory cells are lost, nerves often regress from the sensory area. Therapeutic and regenerative approaches would benefit from the presence of nerve fibers in the tissue. In the hearing system, retraction of afferent innervation may accompany the degeneration of auditory hair cells that is associated with permanent hearing loss. The only therapy currently available for cases with severe or complete loss of hair cells is the cochlear implant auditory prosthesis. To enhance the therapeutic benefits of a cochlear implant, it is necessary to attract nerve fibers back into the cochlear epithelium. Here we show that forced expression of the neurotrophin gene BDNF in epithelial or mesothelial cells that remain in the deaf ear induces robust regrowth of nerve fibers towards the cells that secrete the neurotrophin, and results in re-innervation of the sensory area. The process of neurotrophin-induced neuronal regeneration is accompanied by significant preservation of the spiral ganglion cells. The ability to regrow nerve fibers into the basilar membrane area and protect the auditory nerve will enhance performance of cochlear implants and augment future cell replacement therapies such as stem cell implantation or induced transdifferentiation. This model also provides a general experimental stage for drawing nerve fibers into a tissue devoid of neurons, and studying the interaction between the nerve fibers and the tissue.

A Microassembled Low-Profile Three-Dimensional Microelectrode Array for Neural Prosthesis Applications

This paper describes the design and micro- assembly process of a low-profile 3-D microelectrode array for mapping the functional organization of targeted areas of the central nervous system and for possible application in neural prostheses. The array consists of multiple planar complimentary metal-oxide-semiconductor stimulating probes and 3-D assembly components. Parylene-encapsulated gold beams supported by etch-stopped silicon braces allow the backends of the probes to be folded over to reduce the height of the array above the cortical surface. A process permitting parylene to be used at wafer level with bulk-silicon wet release has been reported. Spacers are used to fix the microassembled probes in position and are equipped with interlocking structures to facilitate the assembly process and increase yield. Four-probe 256-site 3-D arrays operate from plusmn5 V with an average per-channel power dissipation of 97 muW at full range stimulation with pulse widths of 100 mus at 500-Hz frequency. Thirty-two sites can be stimulated simultaneously with maximum currents of plusmn127 muA and a current resolution of plusmn1 muA. The microassembly techniques allow a variety of 3-D microstructures to be created from planar components.

Surgical Implantation and Biocompatibility of Central Nervous System Auditory Prostheses

As part of a program to determine the feasibility of a CNS auditory prosthesis, the tissue reaction to electrodes chronically implanted in the cochlear nucleus (CN) of the guinea pig was examined. Varied open operative approaches and microelectrode designs were utilized. Silicon substrate thin film and platinum-iridium wire electrodes, tethered and untethered, were placed successfully in different divisions of the CN. Implantation through a posterior suboccipital approach was most successful. Histologic examinations demonstrated a glial cell proliferation confined to the area of the electrode track that never exceeded 15 μm in width. No neuronal loss or significant effect on cell morphology was seen, and reactive cells were absent. Electrode migration was apparent in a minority of animal preparations. Although potential problems were identified, our findings lend support to the feasibility of implanting a neuroprosthesis in the CN and have helped to establish methods for future studies of chronic intranuclear stimulation.

A Novel Diamond Microprobe for Neuro-Chemical and -Electrical Recording in Neural Prosthesis

This paper describes the design, microfabrication, and testing of a novel polycrystalline-diamond (poly-C)-based microprobe for possible applications in neural prosthesis. The probe utilizes undoped poly-C with a resistivity on the order of 105 Omega middot cm as a supporting material, which has a Youngs modulus in the range of 400-1000 GPa and is biocompatible. Boron-doped poly-C with a resistivity on the order of 10-3 Omega middot cm is used as an electrode material, which provides a chemically stable surface for both chemical and electrical detections in neural studies. The probe has eight poly-C electrode sites with diameters ranging from 2 to 150 mum; the electrode capacitance is approximately 87 muF/cm2. The measured water potential window of the poly-C electrode spans across negative and positive electrode potentials and typically has a total value of 2.2 V in 1 M KCl. The smallest detectable concentration of norepinephrine (a neurotransmitter) was on the order of 10 nM. The poly-C probe has also been successfully implanted in the auditory cortex area of a guinea pig brain for in vivo neural studies. The recorded signal amplitude was 30-40 muV and had a duration of 1 ms.

Silicon Microelectrodes with Flexible Integrated Cables for Neural Implant Applications

This paper describes two different cable structures that can be integrated with silicon microprobes and fabricated using etch-stops and a wet release at wafer level. One is a serpentine silicon ribbon cable and the other is a parylene cable. They provide highly flexible biocompatible interconnects between the implanted microelectrodes and implanted or external microsystems. Silicon microelectrodes integrated with these cable structures demonstrate consistent and reliable neural recording both acutely and chronically.

Hyaluronic Acid Enhances Gene Delivery into the Cochlea

Cochlear gene therapy can be a new avenue for the treatment of severe hearing loss by inducing regeneration or phenotypic rescue. One necessary step to establish this therapy is the development of a safe and feasible inoculation surgery, ideally without drilling the bony cochlear wall. The round window membrane (RWM) is accessible in the middle-ear space, but viral vectors placed on this membrane do not readily cross the membrane to the cochlear tissues. In an attempt to enhance permeability of the RWM, we applied hyaluronic acid (HA), a nontoxic and biodegradable reagent, onto the RWM of guinea pigs, prior to delivering an adenovirus carrying enhanced green fluorescent protein (eGFP) reporter gene (Ad-eGFP) at the same site. We examined distribution of eGFP in the cochlea 1 week after treatment, comparing delivery of the vector via the RWM, with or without HA, to delivery by a cochleostomy into the perilymph. We found that cochlear tissue treated with HA-assisted delivery of Ad-eGFP demonstrated wider expression of transgenes in cochlear cells than did tissue treated by cochleostomy injection. HA-assisted vector delivery facilitated expression in cells lining the scala media, which are less accessible and not transduced after perilymphatic injection. We assessed auditory function by measuring auditory brainstem responses and determined that thresholds were significantly better in the ears treated with HA-assisted Ad-eGFP placement on the RWM as compared with cochleostomy. Together, these data demonstrate that HA-assisted delivery of viral vectors provides an atraumatic and clinically feasible method to introduce transgenes into cochlear cells, thereby enhancing both research methods and future clinical application.

Effect of electrical pulse shape on AVCN unit responses to cochlear stimulation

Electrical stimulation of the cochlea with a multiple-electrode array is best accomplished using pulsatile instead of continuous stimulation. The optimum shapes of electrical pulses for this purpose are still uncertain due to a lack of knowledge about their stimulation efficiency and requirements of the encoding strategy. We presented an extensive set of charge-balanced, rectangular pulse shapes to the guinea pig cochlea. Durations per phase for these constant-current pulses ranged from 20 microseconds to 900 microseconds with initially positive and initially negative polarities. Spike counts from single units in the anteroventral cochlear nucleus differed significantly for different pulse shapes, as did their initial latencies. Implications for stimulation efficiency and encoding strategies are discussed.