Victor Krauthamer

Containment and growth of neuroblastoma cells on chemically patterned substrates

Patterned substrates offer the promise of controlled positioning and directional guidance of growing neurites. Therefore, they could be useful for constructing small neuronal networks with defined geometry in vitro. We have fabricated chemically patterned substrates using self-assembled monolayer films with a lithographic mask technique and demonstrated the feasibility for geometrically patterning neuroblastoma cells in culture. N-octadecyltrichlorosilane (OTS) was chemically bonded to glass and fused silica substrates, rendering the surface hydrophobic and non-adhesive to cells. Using surface analysis techniques, we have confirmed that OTS films were true monolayers and can be photocleaved from the surface by deep UV irradiation. An adhesive pattern of n-(2-aminoethyl-3-aminopropyl)trimethoxysilane was formed on a selectively irradiated OTS surface via a deep UV lithographic procedure. The chemically patterned surface was then seeded with SK-N-SH human neuroblastoma cells, and cellular attachment and growth were monitored by optical microscopy. The use of 2-dimensional substrates supported the containment and growth of neuroblastoma cells within the pattern for at least 15 days in culture. These chemical patterns may also be useful in controlled arrangements of hearts cells or muscle cells on prosthetic implant devices.

Rapid evaluation of the durability of cortical neural implants using accelerated aging with reactive oxygen species.

OBJECTIVE A challenge for implementing high bandwidth cortical brain-machine interface devices in patients is the limited functional lifespan of implanted recording electrodes. Development of implant technology currently requires extensive non-clinical testing to demonstrate device performance. However, testing the durability of the implants in vivo is time-consuming and expensive. Validated in vitro methodologies may reduce the need for extensive testing in animal models. APPROACH Here we describe an in vitro platform for rapid evaluation of implant stability. We designed a reactive accelerated aging (RAA) protocol that employs elevated temperature and reactive oxygen species (ROS) to create a harsh aging environment. Commercially available microelectrode arrays (MEAs) were placed in a solution of hydrogen peroxide at 87 °C for a period of 7 days. We monitored changes to the implants with scanning electron microscopy and broad spectrum electrochemical impedance spectroscopy (1 Hz-1 MHz) and correlated the physical changes with impedance data to identify markers associated with implant failure. MAIN RESULTS RAA produced a diverse range of effects on the structural integrity and electrochemical properties of electrodes. Temperature and ROS appeared to have different effects on structural elements, with increased temperature causing insulation loss from the electrode microwires, and ROS concentration correlating with tungsten metal dissolution. All array types experienced impedance declines, consistent with published literature showing chronic (>30 days) declines in array impedance in vivo. Impedance change was greatest at frequencies <10 Hz, and smallest at frequencies 1 kHz and above. Though electrode performance is traditionally characterized by impedance at 1 kHz, our results indicate that an impedance change at 1 kHz is not a reliable predictive marker of implant degradation or failure. SIGNIFICANCE ROS, which are known to be present in vivo, can create structural damage and change electrical properties of MEAs. Broad-spectrum electrical impedance spectroscopy demonstrates increased sensitivity to electrode damage compared with single-frequency measurements. RAA can be a useful tool to simulate worst-case in vivo damage resulting from chronic electrode implantation, simplifying the device development lifecycle.

Altered calcium dynamics in cardiac cells grown on silane-modified surfaces

Chemically defined surfaces were created using self-assembled monolayers (SAMs) of hydrophobic and hydrophilic silanes as models for implant coatings, and the morphology and physiology of cardiac myocytes plated on these surfaces were studied in vitro. We focused on changes in intracellular Ca(2+) because of its essential role in regulating heart cell function. The SAM-modified coverslips were analyzed using X-ray Photoelectron Spectroscopy to verify composition. The morphology and physiology of the cardiac cells were examined using fluorescence microscopy and intracellular Ca(2+) imaging. The imaging experiments used the fluorescent ratiometric dye fura-2, AM to establish both the resting Ca(2+) concentration and the dynamic responses to electrical stimulation. A significant difference in excitation-induced Ca(2+) changes on the different silanated surfaces was observed. However, no significant change was noted based on the morphological analysis. This result implies a difference in internal Ca(2+) dynamics, and thus cardiac function, occurs when the composition of the surface is different, and this effect is independent of cellular morphology. This finding has implications for histological examination of tissues surrounding implants, the choice of materials that could be beneficial as implant coatings and understanding of cell-surface interactions in cardiac systems.

Mechanical Bioeffects of Pulsed High Intensity Focused Ultrasound on a Simple Neural Model

PURPOSE To study how pressure pulses affect nerves through mechanisms that are neither thermal nor cavitational, and investigate how the effects are related to cumulative radiation-force impulse (CRFI). Applications include traumatic brain injury and acoustic neuromodulation. METHODS A simple neural model consisting of the giant axon of a live earthworm was exposed to trains of pressure pulses produced by an 825 kHz focused ultrasound transducer. The peak negative pressure of the pulses and duty cycle of the pulse train were controlled so that neither cavitation nor significant temperature rise occurred. The amplitude and conduction velocity of action-potentials triggered in the worm were measured as the magnitude of the pulses and number of pulses in the pulse trains were varied. RESULTS The functionality of the axons decreased when sufficient pulse energy was applied. The level of CRFI at which the observed effects occur is consistent with the lower levels of injury observed in this study relative to blast tubes. The relevant CRFI values are also comparable to CRFI values in other studies showing measureable changes in action-potential amplitudes and velocities. Plotting the measured action-potential amplitudes and conduction velocities from different experiments with widely varying exposure regimens against the single parameter of CRFI yielded values that agreed within 21% in terms of amplitude and 5% in velocity. A predictive model based on the assumption that the temporal rate of decay of action-potential amplitude and velocity is linearly proportional the radiation force experienced by the axon predicted the experimental amplitudes and conduction velocities to within about 20% agreement. CONCLUSIONS The functionality of axons decreased due to noncavitational mechanical effects. The radiation force, possibly by inducing changes in ion-channel permeability, appears to be a possible mechanism for explaining the observed degradation. The CRFI is also a promising parameter for quantifying neural bioeffects during exposure to pressure waves, and for predicting axon functionality.PURPOSE To study how pressure pulses affect nerves through mechanisms that are neither thermal nor cavitational, and investigate how the effects are related to cumulative radiation-force impulse (CRFI). Applications include traumatic brain injury and acoustic neuromodulation. METHODS A simple neural model consisting of the giant axon of a live earthworm was exposed to trains of pressure pulses produced by an 825 kHz focused ultrasound transducer. The peak negative pressure of the pulses and duty cycle of the pulse train were controlled so that neither cavitation nor significant temperature rise occurred. The amplitude and conduction velocity of action-potentials triggered in the worm were measured as the magnitude of the pulses and number of pulses in the pulse trains were varied. RESULTS The functionality of the axons decreased when sufficient pulse energy was applied. The level of CRFI at which the observed effects occur is consistent with the lower levels of injury observed in this study relative to blast tubes. The relevant CRFI values are also comparable to CRFI values in other studies showing measureable changes in action-potential amplitudes and velocities. Plotting the measured action-potential amplitudes and conduction velocities from different experiments with widely varying exposure regimens against the single parameter of CRFI yielded values that agreed within 21% in terms of amplitude and 5% in velocity. A predictive model based on the assumption that the temporal rate of decay of action-potential amplitude and velocity is linearly proportional the radiation force experienced by the axon predicted the experimental amplitudes and conduction velocities to within about 20% agreement. CONCLUSIONS The functionality of axons decreased due to noncavitational mechanical effects. The radiation force, possibly by inducing changes in ion-channel permeability, appears to be a possible mechanism for explaining the observed degradation. The CRFI is also a promising parameter for quantifying neural bioeffects during exposure to pressure waves, and for predicting axon functionality.

Longitudinal vascular dynamics following cranial window and electrode implantation measured with speckle variance optical coherence angiography

Speckle variance optical coherence angiography (OCA) was used to characterize the vascular tissue response from craniotomy, window implantation, and electrode insertion in mouse motor cortex. We observed initial vasodilation ~40% greater than original diameter 2-3 days post-surgery (dps). After 4 weeks, dilation subsided in large vessels (>50 µm diameter) but persisted in smaller vessels (25-50 µm diameter). Neovascularization began 8-12 dps and vessel migration continued throughout the study. Vasodilation and neovascularization were primarily associated with craniotomy and window implantation rather than electrode insertion. Initial evidence of capillary re-mapping in the region surrounding the implanted electrode was manifest in OCA image dissimilarity. Further investigation, including higher resolution imaging, is required to validate the finding. Spontaneous lesions also occurred in many electrode animals, though the inception point appeared random and not directly associated with electrode insertion. OCA allows high resolution, label-free in vivo visualization of neurovascular tissue, which may help determine any biological contribution to chronic electrode signal degradation. Vascular and flow-based biomarkers can aid development of novel neural prostheses.

Excitation and detection of action potential-induced fluorescence changes through a single monomode optical fiber

An optical probe capable of detecting intracellular potential changes in individual cells, in vitro, which has the potential for in vivo applications, has been developed. A single-mode optical fiber directs laser light onto cells stained with the voltage-sensitive fluorescent dye, WW781 and also returns part of the resulting fluorescence to a detection system. Frog cardiac cells in vitro were used in these initial experiments. The fractional change in fluorescent intensity of 10(-3) for a 50 mV shift in transmembrane potential obtained from a heart immobilized in zero calcium Ringers solution is comparable to that reported for other optical methods. For hearts in normal calcium Ringers solutions, very large reproducible motion related artifacts were detected.

BAPTA-AM and ethanol protect cerebellar granule neurons from the destructive effect of the weaver gene.

The mechanisms by which the weaver gene (Reeves et al., 1989; Patil et al., 1995) inhibits neurite extension and/or induces death of the granule neurons in homozygous weaver mouse cerebellum are not presently understood. Here we show that BAPTA‐AM and ethanol, which either reduce cytosolic levels of free calcium or prevent calcium entry, promote neurite outgrowth of the weaver neurons similar to the L‐type calcium channel blocker verapamil (Liesi and Wright, 1996). Importantly, BAPTA‐AM, ethanol, and verapamil not only restore neurite outgrowth of the weaver neurons but adjust their depolarized resting membrane potentials to the levels of normal neurons. These results indicate that calcium‐dependent mechanisms mediate the action of the weaver gene and that the weaver neurons can be normalized by blocking this calcium effect. We further report that BAPTA‐AM and verapamil also have a neuroprotective effect on normal neurons exposed to high concentrations of ethanol. We suggest that verapamil should be evaluated as a drug for treatment of alcohol‐induced brain damage and neurodegenerative disorders. J. Neurosci. Res. 48:571–579, 1997.

Brain–computer interface devices for patients with paralysis and amputation: a meeting report

OBJECTIVE The Food and Drug Administrations (FDA) Center for Devices and Radiological Health (CDRH) believes it is important to help stakeholders (e.g., manufacturers, health-care professionals, patients, patient advocates, academia, and other government agencies) navigate the regulatory landscape for medical devices. For innovative devices involving brain-computer interfaces, this is particularly important. APPROACH Towards this goal, on 21 November, 2014, CDRH held an open public workshop on its White Oak, MD campus with the aim of fostering an open discussion on the scientific and clinical considerations associated with the development of brain-computer interface (BCI) devices, defined for the purposes of this workshop as neuroprostheses that interface with the central or peripheral nervous system to restore lost motor or sensory capabilities. MAIN RESULTS This paper summarizes the presentations and discussions from that workshop. SIGNIFICANCE CDRH plans to use this information to develop regulatory considerations that will promote innovation while maintaining appropriate patient protections. FDA plans to build on advances in regulatory science and input provided in this workshop to develop guidance that provides recommendations for premarket submissions for BCI devices. These proceedings will be a resource for the BCI community during the development of medical devices for consumers.

Use of chemically patterned substrate to study directional effect of damaging electrical stimulation on cultured neuroblastoma cells

We used ordered arrangements of neuroblastoma cells in culture on chemically patterned substrates to direct the orientation of electrical stimulation with respect to cell alignment. Chemically patterned parallel lines of self-assembled monolayer films were fabricated on glass substrates via a deep UV lithographic procedure. Cultured neuroblastoma cells deposited on these substrates formed long (approximately 300 microns in length) neuritic processes along the patterned lines in the presence of retinoic acid. Cells attached to the surface of these substrates were placed in a stimulation chamber so that an electric field (1.4-1.9 V/cm) could be applied in the direction parallel or perpendicular to neuritic or orientation. A majority of cells aligned parallel to the orientation of electrical stimulation exhibited a variety of cellular responses including neuritic tip damage, reductions in neuritic length and varicosity formation. These effects were observed to a lesser degree on the cells when electrical stimulation with the same magnitude was applied perpendicularly to the cell alignment. This work supports earlier findings that geometry is a crucial factor in determining cellular response to applied electric fields and goes on to show that cellular orientation is a key factor in determining cellular damage in culture.

Directional guidance of neurite outgrowth using substrates patterned with biomaterials

The use of geometrically simple networks formed by cultured neurons facilitates the electrophysiological study of biological computation. We used chemically patterned substrates for culturing SK-N-SH human neuroblastoma cells and embryonic rat hippocampal neurons to geometrically control their neurite outgrowth. On patterned substrates (parallel lines, 5-10 microns width), the neuroblastoma cells developed bipolar morphology with long neurite processes (approximately 200 microns) in the presence of retinoic acid. Hippocampal neurons cultured on substrates of hexagonal patterns extended their neurites preferentially along the circumferences of the hexagons and formed geometrically well defined network structures.