In JoVE (1)

Other Publications (31)

Articles by William Shain in JoVE

Other articles by William Shain on PubMed

IEEE Transactions on Neural Systems and Rehabilitation Engineering : a Publication of the IEEE Engineering in Medicine and Biology Society. Jun, 2003  |  Pubmed ID: 12899270

IEEE Transactions on Information Technology in Biomedicine : a Publication of the IEEE Engineering in Medicine and Biology Society. Dec, 2003  |  Pubmed ID: 15000357

IEEE Transactions on Bio-medical Engineering. Sep, 2004  |  Pubmed ID: 15376512

Model Neural Prostheses with Integrated Microfluidics: a Potential Intervention Strategy for Controlling Reactive Cell and Tissue Responses

IEEE Transactions on Bio-medical Engineering. Nov, 2004  |  Pubmed ID: 15536908

Model silicon intracortical probes with microfluidic channels were fabricated and tested to examine the feasibility of using diffusion-mediated delivery to deliver therapeutic agents into the volume of tissue exhibiting reactive responses to implanted devices. Three-dimensional probe structures with microfluidic channels were fabricated using surface micromachining and deep reactive ion etching (DRIE) techniques. In vitro functional tests of devices were performed using fluorescence microscopy to record the transient release of Texas Red labeled transferrin (TR-transferrin) and dextran (TR-dextran) from the microchannels into 1% w/v agarose gel. In vivo performance was characterized by inserting devices loaded with TR-transferrin into the premotor cortex of adult male rats. Brain sections were imaged using confocal microscopy. Diffusion of TR-transferrin into the extracellular space and uptake by cells up to 400 microm from the implantation site was observed in brain slices taken 1 h postinsertion. The reactive tissue volume, as indicated by the presence of phosphorylated mitogen-activated protein kinases (MAPKs), was characterized using immunohistochemistry and confocal microscopy. The reactive tissue volume extended 600, 800, and 400 microm radially from the implantation site at 1 h, 24 h, and 6 weeks following insertion, respectively. These results indicate that diffusion-mediated delivery can be part of an effective intervention strategy for the treatment of reactive tissue responses around chronically implanted intracortical probes.

Lab on a Chip. Jan, 2005  |  Pubmed ID: 15616743

Cytometry. Part A : the Journal of the International Society for Analytical Cytology. Jul, 2005  |  Pubmed ID: 15934061

Synaptic Connectivity of a Low Density Patterned Neuronal Network Produced on the Poly-L-lysine Stamped Microelectrode Array

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. 2005  |  Pubmed ID: 17282041

Rectangular networks of rat hippocampal neurons have been produced on microelectrode arrays (MEAs). The crossing points of networks were located at the recording electrode sites by aligned microcontact printing (μCP) technique. Polydimethysiloxane (PDMS) stamp was fabricated to print fine poly-L-lysine (PLL) patterns of 2 -width lines for neurites and 20 -diameter circles for cell bodies. Different densities of neurons were applied on the PLL-stamped MEAs to find how a low density of neurons still has the functional connectivity. From the neural network applied with a density of 200 cells/mm2, we could observe signal propagation among spontaneous activities. Electrical responses were also evoked by 200 current pulse stimulation with 50 pulse width. Immunocytostaining was employed to identify dendrites, synapses, and nuclei in the patterned neurons.

IEEE Transactions on Neural Systems and Rehabilitation Engineering : a Publication of the IEEE Engineering in Medicine and Biology Society. Jun, 2006  |  Pubmed ID: 16792279

Low-density Neuronal Networks Cultured Using Patterned Poly-l-lysine on Microelectrode Arrays

Journal of Neuroscience Methods. Mar, 2007  |  Pubmed ID: 17049614

Synaptic activity recorded from low-density networks of cultured rat hippocampal neurons was monitored using microelectrode arrays (MEAs). Neuronal networks were patterned with poly-l-lysine (PLL) using microcontact printing (microCP). Polydimethysiloxane (PDMS) stamps were fabricated with relief structures resulting in patterns of 2 microm-wide lines for directing process growth and 20 microm-diameter circles for cell soma attachment. These circles were aligned to electrode sites. Different densities of neurons were plated in order to assess the minimal neuron density required for development of an active network. Spontaneous activity was observed at 10-14 days in networks using neuron densities as low as 200 cells/mm(2). Immunocytochemistry demonstrated the distribution of dendrites along the lines and the location of foci of the presynaptic protein, synaptophysin, on neuron somas and dendrites. Scanning electron microscopy demonstrated that single fluorescent tracks contained multiple processes. Evoked responses of selected portions of the networks were produced by stimulation of specific electrode sites. In addition, the neuronal excitability of the network was increased by the bath application of high K(+) (10-12 mM). Application of DNQX, an AMPA antagonist, blocked all spontaneous activity, suggesting that the activity is excitatory and mediated through glutamate receptors.

Functionalized Hydrogel Surfaces for the Patterning of Multiple Biomolecules

Journal of Biomedical Materials Research. Part A. May, 2007  |  Pubmed ID: 17120223

Patterning of multiple proteins and enzymes onto biocompatible surfaces can provide multiple signals to control cell attachment and growth. Acrylamide-based hydrogels were photo-polymerized in the presence of streptavidin-acrylamide, resulting in planar gel surfaces functionalized with the streptavidin protein. This surface was capable of binding biotin-labeled biomolecules. The proteins fibronectin and laminin, the enzyme alkaline phosphatase, and the photo-protein R-phycoerythrin were patterned using soft lithographic techniques. Polydimethylsiloxane stamps were used to transfer biotinylated proteins onto streptavidin-conjugated hydrogel surfaces. Stamped biomolecules were spatially resolved to feature sizes of 10 mum. Fluorescence measurements were used to assess protein transfer and enzyme functionality on modified surfaces. Our results demonstrate that hydrogel surfaces can be patterned with multiple proteins and enzymes, with retention of biological and catalytic activity. These surfaces are biocompatible and provide cues for cell attachment and growth. (c) 2006 Wiley Periodicals, Inc. J Biomed Mater Res 2007.

Directed Cell Growth on Protein-functionalized Hydrogel Surfaces

Journal of Neuroscience Methods. May, 2007  |  Pubmed ID: 17368788

Biochemical surface modification has been used to direct cell attachment and growth on a biocompatible gel surface. Acrylamide-based hydrogels were photo-polymerized in the presence of an acroyl-streptavidin monomer to create planar, functionalized surfaces capable of binding biotin-labelled proteins. Soft protein lithography (microcontact printing) of proteins was used to transfer the biotinylated extracellular matrix proteins, fibronectin and laminin, and the laminin peptide biotin-IKVAV, onto modified surfaces. As a biological assay, we plated LRM55 astroglioma and primary rat hippocampal neurons on patterned hydrogels. We found both cell types to selectively adhere to areas patterned with biotin-conjugated proteins. Fluorescence and bright-field modes of microscopy were used to assess cell attachment and cell morphology on modified surfaces. LRM55 cells were found to attach to protein-stamped regions of the hydrogel only. Neurons exhibited significant neurite extension after 72h in vitro, and remained viable on protein-stamped areas for more than 4 weeks. Patterned neurons developed functionally active synapses, as measured by uptake of the dye FM1-43FX. Results from this study suggest that hydrogel surfaces can be patterned with multiple proteins to direct cell growth and attachment.

Cytometry. Part A : the Journal of the International Society for Analytical Cytology. Sep, 2007  |  Pubmed ID: 17654650

Electrical Stimulation-induced Cell Clustering in Cultured Neural Networks

Medical & Biological Engineering & Computing. Nov, 2007  |  Pubmed ID: 17684784

Planar microelectrode arrays (MEAs) are widely used to record electrical activity from neural networks. However, only a small number of functional recording sites frequently show electrical activity. One contributing factor may be that neurons in vitro receive insufficient synaptic input to develop into fully functional networks. In this study, electrical stimulation was applied to neurons mimicking synaptic input. Various stimulation paradigms were examined. Stimulation amplitude and frequency were tailored to prevent cell death. Two effects of stimulation were observed when 3-week-old cultures were stimulated: (1) clusters of neural cells were observed adjacent to stimulating electrodes and (2) an increase in spontaneous neuronal activity was recorded at stimulating electrodes. Immunocytochemical analysis indicates that stimulation may cause both new neuron process growth as well as astrocyte activation. These data indicate that electrical stimulation can be used as a tool to modify neural networks at specific electrode sites and promote electrical activity.

Journal of Biomaterials Science. Polymer Edition. 2007  |  Pubmed ID: 17939883

Complex Impedance Spectroscopy for Monitoring Tissue Responses to Inserted Neural Implants

Journal of Neural Engineering. Dec, 2007  |  Pubmed ID: 18057508

A series of animal experiments was conducted to characterize changes in the complex impedance of chronically implanted electrodes in neural tissue. Consistent trends in impedance changes were observed across all animals, characterized as a general increase in the measured impedance magnitude at 1 kHz. Impedance changes reach a peak approximately 7 days post-implant. Reactive responses around individual electrodes were described using immuno- and histo-chemistry and confocal microscopy. These observations were compared to measured impedance changes. Several features of impedance changes were able to differentiate between confined and extensive histological reactions. In general, impedance magnitude at 1 kHz was significantly increased in extensive reactions, starting about 4 days post-implant. Electrodes with extensive reactions also displayed impedance spectra with a characteristic change at high frequencies. This change was manifested in the formation of a semi-circular arc in the Nyquist space, suggestive of increased cellular density in close proximity to the electrode site. These results suggest that changes in impedance spectra are directly influenced by cellular distributions around implanted electrodes over time and that impedance measurements may provide an online assessment of cellular reactions to implanted devices.

Cytometry. Part A : the Journal of the International Society for Analytical Cytology. Jan, 2008  |  Pubmed ID: 18067123

Journal of Neuroscience Methods. May, 2008  |  Pubmed ID: 18294697

Modulation of Cultured Neural Networks Using Neurotrophin Release from Hydrogel-coated Microelectrode Arrays

Journal of Neural Engineering. Jun, 2008  |  Pubmed ID: 18477815

Polyacrylamide and poly(ethylene glycol) diacrylate hydrogels were synthesized and characterized for use as drug release and substrates for neuron cell culture. Protein release kinetics was determined by incorporating bovine serum albumin (BSA) into hydrogels during polymerization. To determine if hydrogel incorporation and release affect bioactivity, alkaline phosphatase was incorporated into hydrogels and a released enzyme activity determined using the fluorescence-based ELF-97 assay. Hydrogels were then used to deliver a brain-derived neurotrophic factor (BDNF) from hydrogels polymerized over planar microelectrode arrays (MEAs). Primary hippocampal neurons were cultured on both control and neurotrophin-containing hydrogel-coated MEAs. The effect of released BDNF on neurite length and process arborization was investigated using automated image analysis. An increased spontaneous activity as a response to the released BDNF was recorded from the neurons cultured on the top of hydrogel layers. These results demonstrate that proteins of biological interest can be incorporated into hydrogels to modulate development and function of cultured neural networks. These results also set the stage for development of hydrogel-coated neural prosthetic devices for local delivery of various biologically active molecules.

Journal of Neuroscience Methods. Aug, 2008  |  Pubmed ID: 18639343

Advanced Neurotechnologies for Chronic Neural Interfaces: New Horizons and Clinical Opportunities

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2008  |  Pubmed ID: 19005048

Biomedical Technologies for in Vitro Screening and Controlled Delivery of Neuroactive Compounds

Central Nervous System Agents in Medicinal Chemistry. 2008  |  Pubmed ID: 19079777

Cell culture models can provide information pertaining to the effective dose, toxiciology, and kinetics, for a variety of neuroactive compounds. However, many in vitro models fail to adequately predict how such compounds will perform in a living organism. At the systems level, interactions between organs can dramatically affect the properties of a compound by alteration of its biological activity or by elimination of it from the body. At the tissue level, interaction between cell types can alter the transport properties of a particular compound, or can buffer its effects on target cells by uptake, processing, or changes in chemical signaling between cells. In any given tissue, cells exist in a three-dimensional environment bounded on all sides by other cells and components of the extracellular matrix, providing kinetics that are dramatically different from the kinetics in traditional two-dimensional cell culture systems. Cell culture analogs are currently being developed to better model the complex transport and processing that occur prior to drug uptake in the CNS, and to predict blood-brain barrier permeability. These approaches utilize microfluidics, hydrogel matrices, and a variety of cell types (including lung epithelial cells, hepatocytes, adipocytes, glial cells, and neurons) to more accurately model drug transport and biological activity. Similar strategies are also being used to control both the spatial and temporal release of therapeutic compounds for targeted treatment of CNS disease.

Biomacromolecules. Jan, 2009  |  Pubmed ID: 19061335

Neuroinformatics. 2009  |  Pubmed ID: 19140032

An in Vitro System for Modeling Brain Reactive Responses and Changes in Neuroprosthetic Device Impedance

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. 2009  |  Pubmed ID: 19965268

Currently available methods for analyzing the structural properties of neural tissue are limited by the frequency at which data can be collected and by the need to sacrifice the specimen to correlate histology with other data. Electrical impedance spectroscopy (EIS) can be used to complement conventional histological and imaging-based methods by measuring real-time electrical data that can be ascribed to changes in tissue composition and structure. This report describes an impedance-based method for the analysis and modeling of the electrical properties of three-dimensional neural tissue constructs in vitro. This model system was used to assess the effects of cell density, type and organization on neuroprosthetic device electrode performance.

IEEE Transactions on Medical Imaging. Mar, 2010  |  Pubmed ID: 20199906

Effects of Glial Cells on Electrode Impedance Recorded from Neuralprosthetic Devices in Vitro

Annals of Biomedical Engineering. Mar, 2010  |  Pubmed ID: 20336824

Neural prosthetic devices hold the potential to be used in the treatment of a variety of neurological disorders. However, their long-term clinical success is currently limited by the ability to achieve stable interfaces between devices and the CNS. Immunohistochemical analysis has shown that cellular responses occur in tissue surrounding implanted devices. These cellular responses have been correlated with the impedance measured from device electrodes, leading to the hypothesis that a possible mechanism resulting in inconsistent device performance is the formation of an electrically insulating glial sheath at the implantation site. However, little is known about what cellular and tissue changes affect impedance values and thus contribute to the decreases in electrode performance. We have designed an in vitro system in which cell conditions can be varied within an artificial tissue matrix surrounding a neural prosthetic device. In this study, high-density cultures of glial cells were analyzed by immunohistochemical methods and impedance spectroscopy. Astrocytes and microglia were cultured at various ratios within the matrix surrounding the probes, and were observed over a period of 2 weeks. Cell seeding conditions and confocal images were compared to impedance data to enable the effects of glial cell type on electrode impedance to be determined.

Optical Monitoring of Neural Networks Evoked by Focal Electrical Stimulation on Microelectrode Arrays Using FM Dyes

Medical & Biological Engineering & Computing. Sep, 2010  |  Pubmed ID: 20490941

Patch-clamping or microelectrode arrays (MEA) are conventional methods to monitor the electrical activity in biological neural networks in vitro. Despite the effectiveness of these techniques, there are disadvantages including the limited number of electrodes and the predetermined location of electrodes in MEAs. In particular, these drawbacks raise a difficulty in monitoring a number of neurons outnumbering the electrodes. Here, we propose an optical technique to determine the effective range of focal electrical stimulation using FM dyes in neural networks grown on planar-type MEAs. After 3 weeks in culture, electrical stimulation was delivered to neural networks via an underlying electrode in the presence of FM dyes. The stimulation induced the internalization of the dye into the neurons around the stimulating electrodes. Fluorescent images of dye distribution successfully showed the effects of focal stimulation. A range of stimulus amplitudes and frequencies were examined to collect fluorescence images. FM-dye uptake after electrical stimulation resulted in the labeling of cells up to approximately 300 microm away from the stimulating electrode. Fluorescence intensity increased proportionally to stimulation amplitude. Tetrodotoxin was shown to inhibit the labeling of neurons except those located immediately adjacent (within 40 microm) from the stimulating electrode. In the presence of AMPA and NMDA receptors antagonists, the FM-dye labeling appeared within 80 microm from the electrode, indicating directly evoked neural networks via blocking of glutamatergic synaptic transmission. These results showed that FM dyes can be a useful tool for monitoring activity-dependent synaptic events and determining the effect of focal stimulation in cultured neural networks.

The Journal of the Acoustical Society of America. Nov, 2010  |  Pubmed ID: 21110568

Acta Biomaterialia. Jun, 2011  |  Pubmed ID: 21345383

Biomaterials. Aug, 2011  |  Pubmed ID: 21609850

Journal of Neuroscience Methods. Jan, 2012  |  Pubmed ID: 21978485

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