Unique electrophysiological property of a novel Nav1.7, Nav1.8, and Nav1.9 sodium channel blocker, ANP-230.

Voltage-gated sodium channel subtypes, Nav1.7, Nav1.8, and Nav1.9 are predominantly expressed in peripheral sensory neurons. Recent genetic studies have revealed that they are involved in pathological pain processing and that the blockade of Nav1.7, Nav1.8, or Nav1.9 will become a promising pharmacotherapy especially for neuropathic pain. A growing number of drug discovery programs have targeted either of the subtypes to obtain a selective inhibitor which can provide pain relief without affecting the cardiovascular and central nervous systems, though none of them has been approved yet. Here we describe the in vitro characteristics of ANP-230, a novel sodium channel blocker under clinical development. Surprisingly, ANP-230 was shown to block three pain-related subtypes, human Nav1.7, Nav1.8, and Nav1.9 with similar potency, but had only low inhibitory activity to human cardiac Nav1.5 channel and rat central Nav channels. The voltage clamp experiments using different step pulse protocols revealed that ANP-230 had a "tonic block" mode of action without state- and use-dependency. In addition, ANP-230 caused a depolarizing shift of the activation curve and decelerated gating kinetics in human Nav1.7-stably expressing cells. The depolarizing shift of activation curve was commonly observed in human Nav1.8-stably expressing cells as well as rat dorsal root ganglion neurons. These data suggested a quite unique mechanism of Nav channel inhibition by ANP-230. Finally, ANP-230 reduced excitability of rat dorsal root ganglion neurons in a concentration dependent manner. Collectively, these promising results indicate that ANP-230 could be a potent drug for neuropathic pain.

"Optical communication with brain cells by means of an implanted duplex micro-device with optogenetics and Ca(2+) fluoroimaging".

To better understand the brain function based on neural activity, a minimally invasive analysis technology in a freely moving animal is necessary. Such technology would provide new knowledge in neuroscience and contribute to regenerative medical techniques and prosthetics care. An application that combines optogenetics for voluntarily stimulating nerves, imaging to visualize neural activity, and a wearable micro-instrument for implantation into the brain could meet the abovementioned demand. To this end, a micro-device that can be applied to the brain less invasively and a system for controlling the device has been newly developed in this study. Since the novel implantable device has dual LEDs and a CMOS image sensor, photostimulation and fluorescence imaging can be performed simultaneously. The device enables bidirectional communication with the brain by means of light. In the present study, the device was evaluated in an in vitro experiment using a new on-chip 3D neuroculture with an extracellular matrix gel and an in vivo experiment involving regenerative medical transplantation and gene delivery to the brain by using both photosensitive channel and fluorescent Ca(2+) indicator. The device succeeded in activating cells locally by selective photostimulation, and the physiological Ca(2+) dynamics of neural cells were visualized simultaneously by fluorescence imaging.

In situ laser micropatterning of proteins for dynamically arranging living cells.

This study shows the modification of the surface of polymer-layered glass substrates to form biofunctional micropatterns through femtosecond laser ablation in an aqueous solution. Domains of micrometer size on a substrate can be selectively converted from proteinphobic (resistant to protein adsorption) to proteinphilic, allowing patterning of protein features under physiological aqueous conditions. When femtosecond laser pulses (800 nm, 1 kHz, 200-500 nJ per pulse) were focused on and scanned on the substrate, which was glass covered with the proteinphobic polymer 2-methacryloyloxyethylphosphorylcholine (MPC), the surface became proteinphilic. Surface analysis by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) reveals that the laser ablates the MPC polymer. Extracellular matrix (ECM) proteins were bound to the laser-ablated surface by physisorption. Since femtosecond laser ablation is induced under physiological aqueous conditions, this approach can form micropatterns of functional ECM proteins with minimal damage. This method was applied to pattern collagen, laminin, and gelatin on the substrate. Removal of an ECM protein from the substrate followed by replacement with another ECM protein was achieved on demand at a specific location and time by the same laser ablation method. Living cells adhered to the fabricated domains where ECM proteins were arranged. The modification of patterning during cell culture was used to control cell migration and form arrays of different cells.

CMOS image sensor integrated with micro-LED and multielectrode arrays for the patterned photostimulation and multichannel recording of neuronal tissue.

We developed a complementary metal oxide semiconductor (CMOS) integrated device for optogenetic applications. This device can interface via neuronal tissue with three functional modalities: imaging, optical stimulation and electrical recording. The CMOS image sensor was fabricated on 0.35 µm standard CMOS process with built-in control circuits for an on-chip blue light-emitting diode (LED) array. The effective imaging area was 2.0 × 1.8 mm². The pixel array was composed of 7.5 × 7.5 µm² 3-transistor active pixel sensors (APSs). The LED array had 10 × 8 micro-LEDs measuring 192 × 225 µm². We integrated the device with a commercial multichannel recording system to make electrical recordings.

Induction of cell-cell connections by using in situ laser lithography on a perfluoroalkyl-coated cultivation platform.

This article describes a novel laser-directed microfabrication method carried out in aqueous solution for the organization of cell networks on a platform. A femtosecond (fs) laser was applied to a platform culturing PC12, HeLa, or normal human astrocyte (NHA) cells to manipulate them and to facilitate mutual connections. By applying an fs-laser-induced impulsive force, cells were detached from their original location on the plate, and translocated onto microfabricated cell-adhesive domains that were surrounded with a cell-repellent perfluoroalkyl (R(f)) polymer. Then the fs-laser pulse-train was applied to the R(f) polymer surface to modify the cell-repellent surface, and to make cell-adhesive channels of several µm in width between each cell-adhesive domain. PC12 cells elongated along the channels and made contact with others cells. HeLa and NHA cells also migrated along the channels and connected to the other cells. Surface analysis by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) confirmed that the R(f) polymer was partially decomposed. The method presented here could contribute not only to the study of developing networks of neuronal, glial, and capillary cells, but also to the quantitative analysis of nerve function.

Morphological evaluation of cell differentiation after the isolation of single cells by a femtosecond laser-induced impulsive force.

When nerve growth factor (NGF) is interacted with PC12 cells derived from rat pheochromocytoma, they are partially differentiated into neuron-like cells with neurites. In this work, PC12 cells differentiated by NGF were selectively isolated using a localized impulsive force in a µm-scale area, which was generated by focusing an infrared femtosecond laser into a cell culture medium. In order to evaluate the ability of the isolation method, differentiated and undifferentiated cells were isolated and their morphological changes after the isolation were compared. In both cases, their neurites were once contracted and some of them gradually regenerated day by day. When differentiated cells were isolated, the percentage of differentiated cells with regenerated neurites, 6 h after the isolation, was about 3.3 times higher than that when undifferentiated ones were isolated. This result was compared with a control trypsin experiment. In the comparison, it was indicated that the same degree of cell function was maintained when the present isolation method was used.