Ultrasound neuromodulation of cultured hippocampal neurons.

Ultrasound is becoming an emerging and promising method for neuromodulation due to its advantage of noninvasiveness and its high spatial resolution. However, the underlying principles of ultrasound neuromodulation have not yet been elucidated. We have herein developed a new in vitro setup to study the ultrasonic neuromodulation, and examined various parameters of ultrasound to verify the effective conditions to evoke the neural activity. Neurons were stimulated with 0.5 MHz center frequency ultrasound, and the action potentials were recorded from rat hippocampal neural cells cultured on microelectrode arrays. As the intensity of ultrasound increased, the neuronal activity also increased. There was a notable and significant increase in both the spike rate and the number of bursts at 50% duty cycle, 1 kHz pulse repetition frequency, and the acoustic intensities of 7.6 W/cm2 and 3.8 W/cm2 in terms of spatial-peak pulse-average intensity and spatial-peak temporal-average intensity, respectively. In addition, the impact of ultrasonic neuromodulation was assessed in the presence of a gamma-aminobutyric acid A (GABAA) receptor antagonist to exclude the effect of activated inhibitory neurons. Interestingly, it is noteworthy that the predominant neuromodulatory effects of ultrasound disappeared when the GABAA blocker was introduced, suggesting the potential of ultrasonic stimulation specifically targeting inhibitory neurons. The experimental setup proposed herein could serve as a useful tool for the clarification of the mechanisms underlying the electrophysiological effects of ultrasound.

Fast Reconfigurable Electrode Array Based on Titanium Oxide for Localized Stimulation of Cultured Neural Network.

Planar microelectrode arrays have become standard tools for in vitro neural-network analysis. However, these predefined micropatterned devices lack adaptability to target-specific cells within a cultured network. Herein, we fabricated a reconfigurable TiO2 electrode array with an anatase-brookite bicrystalline polymorphous mesoporous layer. Because of its selective absorption of ultraviolet (UV) light and corresponding photoconductivity, TiO2 electrode array was identified as a promising tool for high-resolution light-addressing. The TiO2 film was used as a semitransparent semiconductor with a high Roff/Ron ratio of 105 and a fast response time of 400 ms. In addition, the effect of UV radiation on the resistance of the TiO2 film over 30 d in an aqueous environment was analyzed, with the film exhibiting high stability. An arbitrary UV pattern was applied to a reconfigurable TiO2 electrode using a digital micromirror device (DMD), affording highly localized neural stimulation at the single-cell level. The reconfigurable TiO2 electrode with a patterned indium tin oxide (ITO) substrate enabled the independent connection of up to 60 points with external stimulators and signal recorders. We believe this technique would be helpful for electrophysiological research requiring the analysis of cell and neural-network features using a highly localized neural interface.

Co-culture platform for neuron-astrocyte interaction using optogenetic modulation.

For decades, the role of glial cells has attracted attention in the neuroscience field. Particularly, although the astrocyte is the most abundant glial cell type, it was believed to function as a passive support cell. However, recent evidence suggests that astrocytes actively release various gliotransmitters and signaling entities that regulate the excitability of pre-and post-synaptic neurons in the brain. In this study, we optimized the ratio of astrocytes and neurons to investigate the interaction between astrocytes and neurons. To this end, postnatal day 0-1 rodent hippocampi were dissociated and cultured. The neuron-astrocyte ratio was monitored for up to 3 weeks after treating the cultures with 0, 1, and 5 µM of cytosine arabinoside (Ara-C) at DIV 2. Subsequently, from postnatal transgenic (TG) mouse expressing ChR2 on astrocytes, hippocampi were cultured on the microelectrode array (MEA) with the desired neuron-astrocyte ratio. The astrocyte was irradiated using a 473 nm blue laser for 30 s in a cycle of 10 Hz and electrophysiological recording was performed to verify the activities of neurons induced by the stimulated astrocytes. Astrocytes and neurons in both co-cultures increased at an identical ratio when treated with 1 µM Ara-C, whereas they decreased significantly when treated with 5 µM Ara-C. Particularly, the laser-stimulated astrocytes induced an increase in the frequency of neuronal activities and lasted after illumination. The proposed co-culture platform is expected to be used in experiments to investigate the network between astrocytes and neurons in vitro.

Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping.

The evaluation of the protein glycosylation states of samples of aflibercept obtained from three different regions was conducted by site-specific N-linked glycan microheterogeneity profiling. Glycopeptide-based nano-LC MSMS mapping of tryptic-digested samples of each aflibercept lot provided site-specific information about glycan microheterogeneity on each of the five N-glycosylation sites (two sites in the VEGFR-1 region, two sites in the VEGFR-2 region, and one site in the human IgG Fc region). Next, the glycopeptide-mapping results obtained from the three different aflibercept lots were compared to evaluate the similarity between the samples. Three aflibercept lots showed a high degree of similarity in glycan composition, fucosylation level, sialylation level, and branching, when all five N-glycosylation sites were assessed together as a group. On the other hand, noticeable variations between lots in the glycan types and sialylation levels on the two sites of the VEGFR-2 domain were observed when each of the five N-glycosylation sites were assessed using the glycopeptide-based method. The presence of N-glycolylneuraminic acid (NeuGc) glycans, which may mediate adverse immune reactions in antibody therapeutics, were also detected on the sites of VEGFR1 and VEGFR2 domains, but not on the IgG Fc domain site. These results imply that analyses of the glycosylation profiles of fusion proteins containing multiple N-glycosylation sites, such as aflibercept, being done as a part of quality control for the therapeutics manufacturing process or for biosimilar development, can be done with a more applicable outcome by assessing each site separately.

Silicon optrode array with monolithically integrated SU-8 waveguide and single LED light source.

Objective. This paper presents a conventional light emitting diode (LED) and polymer waveguide coupled silicon optrode array.Approach. Unique lens design at the waveguide inlet enables a high light coupling efficiency with a single LED light source, and provides small power consumption compatible with a wireless optogenetic neuromodulation system. To increase the light intensity at the waveguide tip, a lensed waveguide is fabricated with epoxy-based photoresist SU-8, which has a plano-convex lens shape at the waveguide inlet to focus the light in the horizontal direction. In addition, a cylindrical lens is assembled in front of the waveguide inlet to focus the source light in the vertical direction.Main results. The glass cylindrical lens and SU-8 plano-convex lens increased the light coupling efficiency by 6.7 dB and 6.6 dB, respectively. The fabricated 1 × 4 array of optrodes is assembled with a single LED with 465 nm wavelength, which produces a light intensity of approximately 2.7 mW mm-2at the SU-8 waveguide outlet when 50 mA input current is applied to the LED. Each optrode has four recording electrodes at the SU-8 waveguide outlet. The average impedance of the iridium oxide (IrOx) electroplated recording electrodes is 43.6 kΩ.Significance.In-vivoexperiment at the hippocampus region CA1 and CA2 demonstrated the capability of optical stimulation and neural signal recording through the LED and SU-8 waveguide coupled silicon optrode array.

Unusual case of pleural effusion caused by amlodipine in a dog with systemic hypertension.

OBJECTIVE: The aim of this report is to document the case of a dog that developed pleural effusion as a potential side-effect to the administration of a high-dose of amlodipine. CASE SUMMARY: A Yorkshire terrier dog (13-year-old, castrated male, 4.5 kg) presented with severe systemic hypertension (>200 mmHg), hyperkalaemia, and acute pancreatitis. The dog had hyperadrenocorticism, chronic valvular heart disease, chronic kidney disease, and cerebellar infarction as underlying diseases. Additionally, the dog had laboured breathing and tachypnoea during hospitalization. Screening examinations revealed a pleural effusion (pure transudate) for which hypoalbuminemia and thromboembolism were ruled out as the causes. Therefore, the adverse drug event of an anti-hypertensive drug (amlodipine) was tentatively diagnosed. CONCLUSIONS: Pleural effusion resolved within 24 h of reducing the dosage of amlodipine. Hence, the dog was diagnosed with amlodipine-induced pleural effusion. Rarely, amlodipine can cause pleural effusion after high-dose administrations in humans, but only two cases of peripheral edema have been reported in animals. If pleural effusion occurs in hypertensive patients administered amlodipine, it should be considered as the potential cause.

Gene expression of adipokines and inflammatory cytokines in peripheral blood mononuclear cells of obese dogs.

BACKGROUND: Peripheral blood mononuclear cells (PBMCs) have been identified as a possible marker of inflammation in obesity. Understanding the expression of pro- and anti-inflammatory cytokines in PBMCs in obese dogs will help control obesity-related inflammatory diseases. OBJECTIVES: The aim of this study was to evaluate the role of PBMCs in obesity-associated chronic inflammation by analyzing the expression of adipokines and inflammatory cytokines. METHODS: Blood samples were obtained from 25 subjects and real-time quantitative polymerase chain reaction determinations were performed to quantify the gene expression levels of adipokines and inflammatory cytokines, including TNF-α, IL-17, leptin, MCP-1, and adiponectin, in the PBMCs. RESULTS: The results showed that the gene expression levels of TNF-α (p < 0.001), IL-17 (p < 0.0001), and leptin (p < 0.0001) were strongly upregulated in the PBMCs of obese dogs compared to that in non-obese dogs. CONCLUSIONS: The changes in gene expression levels of inflammation-related adipokines and pro-inflammatory cytokines occur in PBMCs, which may contribute to the low-grade chronic inflammation that is present in obesity.

Fabrication of Planar Microelectrode Array Using Laser-Patterned ITO and SU-8.

For several decades, microelectrode array (MEA) has been a powerful tool for in vitro neural electrophysiology because it provides a unique approach for monitoring the activity of a number of neurons over time. Due to the various applications of MEAs with different types of cells and tissues, there is an increasing need to customize the electrode designs. However, the fabrication of conventional MEAs requires several microfabrication procedures of deposition, etching, and photolithography. In this study, we proposed a simple fabrication method with a laser-patterned indium tin oxide (ITO) conductor and SU-8 photoresist insulation. Unlike in a conventional metal patterning process, only the outlines of ITO conductors are ablated by laser without removing background ITO. Insulation is achieved simply via SU-8 photolithography. The electrode sites are electroplated with iridium oxide (IrOX) to improve the electrochemical properties. The fabricated MEAs are electrochemically characterized and the stability of insulation is also confirmed by impedance monitoring for three weeks. Dissociated neurons of rat hippocampi are cultured on MEAs to verify the biocompatibility and the capacity for extracellular neural recording. The electrochemical and electrophysiological results with the fabricated MEAs are similar to those from conventional SiNX-insulated MEAs. Therefore, the proposed MEA with laser-patterned ITO and SU-8 is cost-effective and equivalently feasible compared with the conventional MEAs fabricated using thin-film microfabrication techniques.

Autoimmune polyendocrine syndrome with hypoadrenocorticism and diabetes mellitus in a dog: A rare case.

BACKGROUND: Autoimmune polyendocrine syndrome, also called polyglandular autoimmune syndrome, is a rare immune-mediated disorder that involves various endocrine glands. PURPOSE: To report autoimmune polyendocrine syndrome in a dog. METHODS: A 9-year-old spayed female miniature poodle diagnosed with insulin-dependent diabetes mellitus emergently visited our clinic for anorexia, severe depression, and vomiting. Hyponatremia, hypochloridemia, and recurrent hypoglycaemia were found. Hypoadrenocorticism was diagnosed based on consistent clinical signs and repeated adrenocorticotropic hormone stimulation tests. RESULTS: After injecting deoxycorticosterone pivalate and increasing the oral prednisolone dose, the patient's systemic condition improved. CONCLUSIONS: To the best of our knowledge, this is the first case report of hypoadrenocorticism concurrent with diabetes mellitus in a dog. Furthermore, we would like to present the probability of an immune-mediated disorder with multiple organs involved, like type IV autoimmune polyendocrine syndrome in humans.

Preparing cuprous oxide nanomaterials by electrochemical method for non-enzymatic glucose biosensor.

Cuprous oxide (Cu2O) nanostructure has been synthesized using an electrochemical method with a two-electrode system. Cu foils were used as electrodes and NH2(OH) was utilized as the reducing agent. The effects of pH and applied voltages on the morphology of the product were investigated. The morphology and optical properties of Cu2O particles were characterized using scanning electron microscopy, x-ray diffraction, and diffuse reflectance spectra. The synthesized Cu2O nanostructures that formed in the vicinity of the anode at 2 V and pH = 11 showed high uniform distribution, small size, and good electrochemical sensing. These Cu2O nanoparticles were coated on an Indium tin oxide substrate and applied to detect non-enzyme glucose as excellent biosensors. The non-enzyme glucose biosensors exhibited good performance with high response, good selectivity, wide linear detection range, and a low detection limit at 0.4 µM. Synthesized Cu2O nanostructures are potential materials for a non-enzyme glucose biosensor.

Photothermal activation of astrocyte cells using localized surface plasmon resonance of gold nanorods.

Although it has been revealed that astrocytes, generally known as star-shaped glial cells, play critical roles in the functions of central nervous system, there have been few efforts to directly modulate their activities and responses. In this study, an optical stimulation strategy for producing intracellular Ca2+ transients of astrocytes is demonstrated using near-infrared (NIR) light and localized surface plasmon resonance. It is presented that NIR stimulation of micro-second duration combined with gold nanorods (GNRs) efficiently produces stronger Ca2+ transients of astrocytes, which seems to be associated with a local heat generation by photothermal effects of GNRs. Since the proposed scheme can directly activate astrocytes with a high reliability, it is expected that GNR-mediated NIR stimulation could be utilized to facilitate minimally invasive physiological studies on the astrocyte functions. Photos of intracellular Ca2+ transient of astrocytes with membrane-bound GNRs after optical stimulation at 30 s.

Synergistic combination of near-infrared irradiation and targeted gold nanoheaters for enhanced photothermal neural stimulation.

Despite a potential of infrared neural stimulation (INS) for modulating neural activities, INS suffers from limited light confinement and bulk tissue heating. Here, a novel methodology for an advanced optical stimulation is proposed by combining near-infrared (NIR) stimulation with gold nanorods (GNRs) targeted to neuronal cell membrane. We confirmed experimentally that in vitro and in vivo neural activation is associated with a local heat generation based on NIR stimulation and GNRs. Compared with the case of NIR stimulation without an aid of GNRs, combination with cell-targeted GNRs allows photothermal stimulation with faster neural response, lower delivered energy, higher stimulation efficiency and stronger behavior change. Since the suggested method can reduce a requisite radiant exposure level and alleviate a concern of tissue damage, it is expected to open up new possibilities for applications to optical neuromodulations for diverse excitable tissues and treatments of neurological disorders.

Multi-color fluorescence imaging based on plasmonic wavelength selection and double illumination by white light.

We demonstrate the proof-of-concept for developing a multi-color fluorescence imaging system based on plasmonic wavelength selection and double illumination by white light source. This technique is associated with fluorescence excitation by transmitted light via a diffraction of propagating surface plasmons. Since double illumination through both sides of isosceles triangle prism in the Kretschmann configuration enables multiple transmission beams of different wavelengths to interact with the specimen, our approach can be an alternative to conventional fluorescence detection owing to alignment stability and functional expandability. After fabricating a plasmonic wavelength splitter and integrating it with microscopic imaging system, we successfully confirm the performance by visualizing in vitro neuron cells labeled with green and red fluorescence dyes. The suggested method has a potential that it could be combined with plasmonic biosensor scheme to realize a multi-functional platform which allows imaging and sensing of biological samples at the same time.