Myocardial ischemia-reperfusion arrhythmia after cardiac surgery is common and seriously affects quality of life. Remote ischemic preconditioning can reduce the myocardial damage caused by severe ischemia. However, the underlying mechanism is not well understood. This study aimed to investigate the effects of exosomes derived from C2C12 mouse myoblasts after hypoxic preconditioning (HP) on ventricular conduction in hypothermic ischemia-reperfusion hearts. Myocardial ischemia-reperfusion model rats were established using the Langendorff cardiac perfusion system. Exosomes derived from normoxic (ExoA) and hypoxia-preconditioned (ExoB) C2C12 cells were injected into the jugular vein of the model rats. The time to heartbeat restoration, arrhythmia type and duration, and heart rate were recorded after myocardial ischemia-reperfusion. Conduction velocity on the surface of left ventricle was measured using a microelectrode array after 30 min of balanced perfusion, 15 min of reperfusion, and 30 min of reperfusion. Immunohistochemistry and western blotting were performed to determine the distribution and relative expression of connexin 43 (Cx43). ExoB contained more exosomes than ExoA, showing that HP stimulated the release of exosomes. The IR + ExoB group showed faster recovery of ventricular myocardial activity, a lower arrhythmia score, faster conduction velocity, and better electrical conductivity than the IR group. ExoB increased the expression of Cx43 and reduced its lateralization in the ventricular muscle. Our study showed that exosomes induced by hypoxic preconditioning can improve ventricular myocardial conduction and reperfusion arrhythmia in isolated hearts after hypothermic ischemia-reperfusion.
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Brain activity implies the orchestrated functioning of interconnected brain regions. Typical in vitro models aim to mimic the brain using single human pluripotent stem cell-derived neuronal networks. However, the field is constantly evolving to model brain functions more accurately through the use of new paradigms, e.g., brain-on-a-chip models with compartmentalized structures and integrated sensors. These methods create novel data requiring more complex analysis approaches. The previously introduced circular tripartite network concept models the connectivity between spatially diverse neuronal structures. The model consists of a microfluidic device allowing axonal connectivity between separated neuronal networks with an embedded microelectrode array to record both local and global electrophysiological activity patterns in the closed circuitry. The existing tools are suboptimal for the analysis of the data produced with this model. Here, we introduce advanced tools for synchronization and functional connectivity assessment. We used our custom-designed analysis to assess the interrelations between the kainic acid (KA)-exposed proximal compartment and its nonexposed distal neighbors before and after KA. Novel multilevel circuitry bursting patterns were detected and analyzed in parallel with the inter- and intracompartmental functional connectivity. The effect of KA on the proximal compartment was captured, and the spread of this effect to the nonexposed distal compartments was revealed. KA induced divergent changes in bursting behaviors, which may be explained by distinct baseline activity and varied intra- and intercompartmental connectivity strengths. The circular tripartite network concept combined with our developed analysis advances importantly both face and construct validity in modeling human epilepsy in vitro.

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the adult brain that binds to GABA receptors and hyperpolarizes the postsynaptic neuron. Gabazine acts as a competitive antagonist to type A GABA receptors (GABAAR), thereby causing diminished neuronal hyperpolarization and GABAAR-mediated inhibition. However, the biochemical effects and the potential regulatory role of astrocytes in this process remain poorly understood. To address this, we investigated the neuronal responses of gabazine in rat cortical cultures containing varying ratios of neurons and astrocytes. Electrophysiological characterization was performed utilizing microelectrode arrays (MEAs) with topologically controlled microcircuit cultures that enabled control of neuronal network growth. Biochemical analysis of the cultures was performed using traditional dissociated cultures on coverslips. Our study indicates that, upon gabazine stimulation, astrocyte-rich neuronal cultures exhibit elevated electrophysiological activity and tyrosine phosphorylation of tropomyosin receptor kinase B (TrkB; receptor for brain-derived neurotrophic factor), along with distinct cytokine secretion profiles. Notably, neurons lacking proper astrocytic support were found to experience synapse loss and decreased mitogen-activated protein kinase (MAPK) phosphorylation. Furthermore, astrocytes contributed to neuronal viability, morphology, vascular endothelial growth factor (VEGF) secretion, and overall neuronal network functionality, highlighting the multifunctional role of astrocytes.

HIV-associated neurocognitive disorders (HAND) persist under antiretroviral therapy as a complex pathology that has been difficult to study in cellular and animal models. Therefore, we generated an ex vivo human brain slice model of HIV-1 infection from surgically resected adult brain tissue. Brain slice cultures processed for flow cytometry showed >90% viability of dissociated cells within the first three weeks in vitro, with parallel detection of astrocyte, myeloid, and neuronal populations. Neurons within brain slices showed stable dendritic spine density and mature spine morphologies in the first weeks in culture, and they generated detectable activity in multi-electrode arrays. We infected cultured brain slices using patient-matched CD4+ T-cells or monocyte-derived macrophages (MDMs) that were exposed to a GFP-expressing R5-tropic HIV-1 in vitro. Infected slice cultures expressed viral RNA and developed a spreading infection up to 9 days post-infection, which were significantly decreased by antiretrovirals. We also detected infected myeloid cells and astrocytes within slices and observed minimal effect on cellular viability over time. Overall, this human-centered model offers a promising resource to study the cellular mechanisms contributing to HAND (including antiretroviral toxicity, substance use, and aging), infection of resident brain cells, and new neuroprotective therapeutics.

BACKGROUND: Astrocytes are the most abundant cell type of the central nervous system and are fundamentally involved in homeostasis, neuroprotection, and synaptic plasticity. This regulatory function of astrocytes on their neighboring cells in the healthy brain is subject of current research. In the ischemic brain we assume disease specific differences in astrocytic acting. The renin-angiotensin-aldosterone system regulates arterial blood pressure through endothelial cells and perivascular musculature. Moreover, astrocytes express angiotensin II type 1 and 2 receptors. However, their role in astrocytic function has not yet been fully elucidated. We hypothesized that the angiotensin II receptors impact astrocyte function as revealed in an in vitro system mimicking cerebral ischemia. Astrocytes derived from neonatal wistar rats were exposed to telmisartan (angiotensin II type 1 receptor-blocker) or PD123319 (angiotensin II type 2 receptor-blocker) under normal conditions (control) or deprivation from oxygen and glucose. Conditioned medium (CM) of astrocytes was harvested to elucidate astrocyte-mediated indirect effects on microglia and cortical neurons.
RESULTS: The blockade of angiotensin II type 1 receptor by telmisartan increased the survival of astrocytes during ischemic conditions in vitro without affecting their proliferation rate or disturbing their expression of S100A10, a marker of activation. The inhibition of the angiotensin II type 2 receptor pathway by PD123319 resulted in both increased expression of S100A10 and proliferation rate. The CM of telmisartan-treated astrocytes reduced the expression of pro-inflammatory mediators with simultaneous increase of anti-inflammatory markers in microglia. Increased neuronal activity was observed after treatment of neurons with CM of telmisartan- as well as PD123319-stimulated astrocytes.
CONCLUSIONS: Data show that angiotensin II receptors have functional relevance for astrocytes that differs in healthy and ischemic conditions and effects surrounding microglia and neuronal activity via secretory signals. Above that, this work emphasizes the strong interference of the different cells in the CNS and that targeting astrocytes might serve as a therapeutic strategy to influence the acting of glia-neuronal network in de- and regenerative context.

In recent years, in vitro three-dimensional (3D) neuronal network models utilizing extracellular matrices have been advancing. To understand the network activity from these models, attempts have been made to measure activity in multiple regions simultaneously using a microelectrode array (MEA). Although there hve been many attempts to measure the activity of 3D networks using 2-dimensional (2D) MEAs, the physical coupling between the 3D network and the microelectrodes was not stable and needed to be improved. In this study, we proposed a neuronal cluster interface that improves the active channel ratio of commercial 2D MEAs, enabling reliable measurement of 3D network activity. To achieve this, neuronal clusters, which consist of a small number of neurons, were patterned on microelectrodes and used as mediators to transmit the signal between the 3D network and the microelectrodes. We confirmed that the patterned neuronal clusters enhanced the active channel ratio and SNR(signal-to-noise-ratio) about 3D network recording and stimulation for a month. Our interface was able to functionally connect with 3D networks and measure the 3D network activity without significant alternation of activity characteristics. Finally, we demonstrated that our interface can be used to analyze the differences in the dynamics of 3D and 2D networks and to construct the 3D clustered network. This method is expected to be useful for studying the functional activity of various 3D neuronal network models, offering broad applications for the use of these models.

UNASSIGNED: Pancreatic islets are important in nutrient homeostasis and improved cellular models of clonal origin may very useful especially in view of relatively scarce primary material. Close 3D contact and coupling between β-cells are a hallmark of physiological function improving signal/noise ratios. Extracellular electrophysiology using micro-electrode arrays (MEA) is technically far more accessible than single cell patch clamp, enables dynamic monitoring of electrical activity in 3D organoids and recorded multicellular slow potentials (SP) provide unbiased insight in cell-cell coupling.
UNASSIGNED: We have therefore asked whether 3D spheroids enhance clonal β-cell function such as electrical activity and hormone secretion using human EndoC-βH1, EndoC-βH5 and rodent INS-1 832/13 cells.
UNASSIGNED: Spheroids were formed either by hanging drop or proprietary devices. Extracellular electrophysiology was conducted using multi-electrode arrays with appropriate signal extraction and hormone secretion measured by ELISA.
UNASSIGNED: EndoC-βH1 spheroids exhibited increased signals in terms of SP frequency and especially amplitude as compared to monolayers and even single cell action potentials (AP) were quantifiable. Enhanced electrical signature in spheroids was accompanied by an increase in the glucose stimulated insulin secretion index. EndoC-βH5 monolayers and spheroids gave electrophysiological profiles similar to EndoC-βH1, except for a higher electrical activity at 3 mM glucose, and exhibited moreover a biphasic profile. Again, physiological concentrations of GLP-1 increased AP frequency. Spheroids also exhibited a higher secretion index. INS-1 cells did not form stable spheroids, but overexpression of connexin 36, required for cell-cell coupling, increased glucose responsiveness, dampened basal activity and consequently augmented the stimulation index.
UNASSIGNED: In conclusion, spheroid formation enhances physiological function of the human clonal β-cell lines and these models may provide surrogates for primary islets in extracellular electrophysiology.

Electroplating of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) is important in many neuroelectronic applications but is challenging to achieve uniformity on large-scale microelectrode arrays (MEA) using conventional galvanostatic methods. In this study, we address this challenge through a potentiostatic method and demonstrate highly uniform electroplating of PEDOT:PSS on MEA with more than one hundred electrodes, all at cellular sizes. The validation of this approach involves comparisons with galvanostatic deposition methods, showcasing unparalleled deposition yield and uniformity. Systematic electrochemical characterizations reveal similarities in structure and stability from potentiostatic deposited coatings. The advances developed here establish the potentiostatic method and detailed process to achieve a uniform coating of PEDOT:PSS on large-scale MEA, with broad utility in neuroelectronics.

Multichannel arrays capable of real-time sensing of neuromodulators in the brain are crucial for gaining insights into new aspects of neural communication. However, measuring neurochemicals, such as dopamine, at low concentrations over large areas has proven challenging. In this research, we demonstrate a novel approach that leverages the scalability and processing power offered by microelectrode array devices integrated with a functionalized, high-density microwire bundle, enabling electrochemical sensing at an unprecedented scale and spatial resolution. The sensors demonstrate outstanding selective molecular recognition by incorporating a selective polymeric membrane. By combining cutting-edge commercial multiplexing, digitization, and data acquisition hardware with a bio-compatible and highly sensitive neurochemical interface array, we establish a powerful platform for neurochemical analysis. This multichannel array has been successfully utilized in vitro and ex vivo systems. Notably, our results show a sensing area of 2.25 mm2 with an impressive detection limit of 820 pM for dopamine. This new approach paves the way for investigating complex neurochemical processes and holds promise for advancing our understanding of brain function and neurological disorders.

In this paper, we report a low-cost printing process of carbon nanotube (CNT)-based, all-organic microelectrode arrays (MEAs) suitable for in vitro neural stimulation and recording. Conventional MEAs have been mainly composed of expensive metals and manufactured through high-cost and complex lithographic processes, which have limited their accessibility for neuroscience experiments and their application in various studies. Here, we demonstrate a printing-based fabrication method for microelectrodes using organic CNT/paraffin ink, coupled with the deposition of an insulating layer featuring single-cell-sized sensing apertures. The simple microfabrication processes utilizing the economic and readily available ink offer potential for cost reduction and improved accessibility of MEAs. Biocompatibility of the fabricated microelectrode was suggested through a live/dead assay of cultured neural cells, and its large electric double layer capacitance was revealed by cyclic voltammetry that was crucial for preventing cytotoxic electrolysis during electric neural stimulation. Furthermore, the electrode exhibited sufficiently low electric impedance of 2.49 Ω·cm2 for high signal-to-noise ratio neural recording, and successfully captured model electric waves in physiological saline solution. These results suggest the easily producible and low-cost printed all-organic microelectrodes are available for neural stimulation and recording, and we believe that they can expand the application of MEA in various neuroscience research.