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Astrocytes are responsible for maintaining homoeostasis and cognitive functions through calcium signalling, a process that is altered in brain diseases. Current bioelectronic tools are designed to study neurons and are not suitable for controlling calcium signals in astrocytes. Here, we show that electrical stimulation of astrocytes using electrodes coated with graphene oxide and reduced graphene oxide induces respectively a slow response to calcium, mediated by external calcium influx, and a sharp one, exclusively due to calcium release from intracellular stores. Our results suggest that the different conductivities of the substrate influence the electric field at the cell-electrolyte or cell-material interfaces, favouring different signalling events in vitro and ex vivo. Patch-clamp, voltage-sensitive dye and calcium imaging data support the proposed model. In summary, we provide evidence of a simple tool to selectively control distinct calcium signals in brain astrocytes for straightforward investigations in neuroscience and bioelectronic medicine., (© 2024. The Author(s).)

The application of light in studying and influencing cellular behavior with improved temporal and spatial resolution remains a key objective in fields such as chemistry, physics, medicine, and engineering. In the brain, nonexcitable cells called astrocytes play essential roles in regulating homeostasis and cognitive function through complex calcium signaling pathways. Understanding these pathways is vital for deciphering brain physiology and neurological disorders like Parkinson's and Alzheimer's. Despite challenges in selectively targeting astrocyte signaling pathways due to shared molecular equipment with neurons, recent advancements in laser technology offer promising avenues. However, the effort to use laser light properties to study astroglial cell function is still limited. This work aims to exploit an in-depth pharmacological analysis of astrocyte calcium channels to determine the physiological mechanism induced by exposure to classical nanosecond-pulsed light. We herein report molecular clues supporting the use of visible-nanosecond laser pulses as a promising approach to excite primary rat neocortical astrocytes and unprecedentedly report on the implementation of entangled two-photon microscopy to image them.

In the adult brain, the water channel aquaporin-4 (AQP4) is expressed in astrocyte endfoot, in supramolecular assemblies, called "Orthogonal Arrays of Particles" (OAPs) together with the transient receptor potential vanilloid 4 (TRPV4), finely regulating the cell volume. The present study aimed at investigating the contribution of AQP4 and TRPV4 to CNS early postnatal development using WT and AQP4 KO brain and retina and neuronal stem cells (NSCs), as an in vitro model of astrocyte differentiation. Western blot analysis showed that, differently from AQP4 and the glial cell markers, TRPV4 was downregulated during CNS development and NSC differentiation. Blue native/SDS-PAGE revealed that AQP4 progressively organized into OAPs throughout the entire differentiation process. Fluorescence quenching assay indicated that the speed of cell volume changes was time-related to NSC differentiation and functional to their migratory ability. Calcium imaging showed that the amplitude of TRPV4 Ca 2+ transient is lower, and the dynamics are changed during differentiation and suppressed in AQP4 KO NSCs. Overall, these findings suggest that early postnatal neurodevelopment is subjected to temporally modulated water and Ca 2+ dynamics likely to be those sustaining the biochemical and physiological mechanisms responsible for astrocyte differentiation during brain and retinal development., (© 2024 The Authors. GLIA published by Wiley Periodicals LLC.)

The increasing prevalence of chronic diseases is a driver for emerging big data technologies for healthcare including digital platforms for data collection, systems for active patient engagement and education, therapy specific predictive models, optimized patient pathway models. Powerful bioelectronic medicine tools for data collection, analysis and visualization allow for joint processing of large volumes of heterogeneous data, which in turn can produce new insights about patient outcomes and alternative interpretations of clinical patterns that can lead to implementation of optimized clinical decisions and clinical patient pathway by healthcare professionals.With this perspective, we identify innovative solutions for disease management and evaluate their impact on patients, payers and society, by analyzing their impact in terms of clinical outcomes (effectiveness, safety, and quality of life) and economic outcomes (cost-effectiveness, savings, and productivity).As a result, we propose a new approach based on the main pillars of innovation in the disease management area, i.e. progressive patient care models, patient-centric approaches, bioelectronics for precise medicine, and lean management that, combined with an increase in appropriate private-public-citizen-partnership, leads towards Patient-Centric Healthcare., (© 2023. The Author(s).)

Despite of the major role of aquaporin (AQP) water channels in controlling transmembrane water fluxes, alternative ways for modulating water permeation have been proposed. In the Central Nervous System (CNS), Aquaporin-4 (AQP4) is reported to be functionally coupled with the calcium-channel Transient-Receptor Potential Vanilloid member-4 (TRPV4), which is controversially involved in cell volume regulation mechanisms and water transport dynamics. The present work aims to investigate the selective role of TRPV4 in regulating plasma membrane water permeability in an AQP4-independent way. Fluorescence-quenching water transport experiments in Aqp4 -/- astrocytes revealed that cell swelling rate is significantly increased upon TRPV4 activation and in the absence of AQP4. The biophysical properties of TRPV4-dependent water transport were therefore assessed using the HEK-293 cell model. Calcein quenching experiments showed that chemical and thermal activation of TRPV4 overexpressed in HEK-293 cells leads to faster swelling kinetics. Stopped-flow light scattering water transport assay was used to measure the osmotic permeability coefficient ( Pf , cm/s) and activation energy ( Ea , kcal/mol) conferred by TRPV4. Results provided evidence that although the Pf measured upon TRPV4 activation is lower than the one obtained in AQP4-overexpressing cells ( Pf of AQP4 = 0.01667 ± 0.0007; Pf of TRPV4 = 0.002261 ± 0.0004; Pf of TRPV4 + 4αPDD = 0.007985 ± 0.0006; Pf of WT = 0.002249 ± 0.0002), along with activation energy values ( Ea of AQP4 = 0.86 ± 0.0006; Ea of TRPV4 + 4αPDD = 2.73 ± 1.9; Ea of WT = 8.532 ± 0.4), these parameters were compatible with a facilitated pathway for water movement rather than simple diffusion. The possibility to tune plasma membrane water permeability more finely through TRPV4 might represent a protective mechanism in cells constantly facing severe osmotic challenges to avoid the potential deleterious effects of the rapid cell swelling occurring via AQP channels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Barile, Mola, Formaggio, Saracino, Cibelli, Gargano, Mogni, Frigeri, Caprini, Benfenati and Nicchia.)

Astrocytes are key regulators of brain homeostasis, equilibrating ion, water, and neurotransmitter concentrations and maintaining essential conditions for proper cognitive function. Recently, it has been shown that the excitability of the actin cytoskeleton manifests in second-scale dynamic fluctuations and acts as a sensor of chemophysical environmental cues. However, it is not known whether the cytoskeleton is excitable in astrocytes and how the homeostatic function of astrocytes is linked to the dynamics of the cytoskeleton. Here it is shown that homeostatic regulation involves the excitable dynamics of actin in certain subcellular regions of astrocytes, especially near the cell boundary. The results further indicate that actin dynamics concentrate into "hotspot" regions that selectively respond to certain chemophysical stimuli, specifically the homeostatic challenges of ion or water concentration increases. Substrate topography makes the actin dynamics of astrocytes weaker. Super-resolution images demonstrate that surface topography is also associated with the predominant perpendicular alignment of actin filaments near the cell boundary, whereas flat substrates result in an actin cortex mainly parallel to the cell boundary. Additionally, coculture with neurons increases both the probability of actin dynamics and the strength of hotspots. The excitable systems character of actin thus makes astrocytes direct participants in neural cell network dynamics., (© 2023 Wiley-VCH GmbH.)

Interoception is the process by which the nervous system regulates internal functions to achieve homeostasis. The role of neurons in interoception has received considerable recent attention, but glial cells also contribute. Glial cells can sense and transduce signals including osmotic, chemical, and mechanical status of extracellular milieu. Their ability to dynamically communicate "listening" and "talking" to neurons is necessary to monitor and regulate homeostasis and information integration in the nervous system. This review introduces the concept of "Glioception" and focuses on the process by which glial cells sense, interpret and integrate information about the inner state of the organism. Glial cells are ideally positioned to act as sensors and integrators of diverse interoceptive signals and can trigger regulatory responses via modulation of the activity of neuronal networks, both in physiological and pathological conditions. We believe that understanding and manipulating glioceptive processes and underlying molecular mechanisms provide a key path to develop new therapies for the prevention and alleviation of devastating interoceptive dysfunctions, among which pain is emphasized here with more focused details., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

This work describes the development of electroconductive hydrogels as injectable matrices for neural tissue regeneration by exploiting a biocompatible conductive polymer - poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) - combined with a biomimetic polymer network made of gelatin. Our approach involved also genipin - a natural cross-linking agent - to promote gelation of gelatin networks embedding PEDOT:PSS. The achieved results suggest that physical-chemical properties of the resulting hydrogels, like impedance, gelation time, mechanical properties, swelling and degradation in physiological conditions, can be finely tuned by the amount of PEDOT:PSS and genipin used in the formulation. Furthermore, the presence of PEDOT:PSS (i) enhances the electrical conductivity, (ii) improves the shear modulus of the resulting hydrogels though (iii) partially impairing their resistance to shear deformation, (iv) reduces gelation time and (v) reduces their swelling ability in physiological medium. Additionally, the resulting electroconductive hydrogels demonstrate enhanced adhesion and growth of primary rat cortical astrocytes. Given the permissive interaction of hydrogels with primary astrocytes, the presented biomimetic, electroconductive and injectable hydrogels display potential applications as minimally invasive systems for neurological therapies and damaged brain tissue repair.

Background/aims: The ability of astrocytes to control extracellular volume homeostasis is critical for brain function and pathology. Uncovering the mechanisms of cell volume regulation by astrocytes will be important for identifying novel therapeutic targets for neurological conditions, such as those characterized by imbalances to hydro saline challenges (as in edema) or by altered cell volume regulation (as in glioma). One major challenge in studying the astroglial membrane channels involved in volume homeostasis in cell culture model systems is that the expression patterns of these membrane channels do not resemble those observed in vivo. In our previous study, we demonstrated that rat primary astrocytes grown on nanostructured interfaces based on hydrotalcite-like compounds (HTlc) in vitro are differentiated and display molecular and functional properties of in vivo astrocytes, such as the functional expression of inwardly rectifying K + channel (Kir 4.1) and Aquaporin-4 (AQP4) at the astrocytic microdomain. Here, we take advantage of the properties of differentiated primary astrocytes in vitro to provide an insight into the mechanism underpinning astrocytic cell volume regulation and its correlation with the expression and function of AQP4, Transient Receptor Potential Vanilloid 4
(TRPV4), and Volume Regulated Anion Channel (VRAC)., Methods: The calcein quenching method was used to study water transport and cell volume regulation. Calcium imaging and electrophysiology (patch-clamp) were used for functional analyses of calcium dynamics and chloride currents. Western blot and immunofluorescence were used to analyse the expression and localization of the channel proteins of interest., Results: We found that the increase in water permeability, previously observed in differentiated astrocytes, occurs simultaneously with more efficient regulatory volume increase and regulatory volume decrease. Accordingly, the magnitude of the hypotonic induced intracellular calcium response, typically mediated by TRPV4, as well as the hypotonic induced VRAC current, was almost twice as high in differentiated astrocytes. Interestingly, while we confirmed increased AQP4 expression in the membrane of differentiated astrocytes, the expression of the channels TRPV4 and Leucine-Rich Repeats-Containing 8-A (LRRC8-A) were comparable between differentiated and non-differentiated astrocytes., Conclusion: The reported results indicate that AQP4 up-regulation observed in differentiated astrocytes might promote higher sensitivity of the cell to osmotic changes, resulting in increased magnitude of calcium signaling and faster kinetics of the RVD and RVI processes. The implications for cell physiology and the mechanisms underlying astrocytic interaction with nanostructured interfaces are discussed., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Recent studies have proposed that the bioelectrical response of glial cells, called astrocytes, currently represents a key target for neuroregenerative purposes. Here, we propose the fabrication of electrospun nanofibres containing gelatin and polyaniline (PANi) synthesized in the form of nano-needles (PnNs) as electrically conductive scaffolds to support the growth and functionalities of primary astrocytes. We report a fine control of the morphological features in terms of fibre size and spatial distribution and fibre patterning, i.e. random or aligned fibre organization, as revealed by SEM- and TEM-supported image analysis. We demonstrate that the peculiar morphological properties of fibres - i.e. , the fibre size scale and alignment - drive the adhesion, proliferation, and functional properties of primary cortical astrocytes. In addition, the gradual transmission of biochemical and biophysical signals due to the presence of PnNs combined with the presence of gelatin results in a permissive and guiding environment for astrocytes. Accordingly, the functional properties of astrocytes measured via cell patch-clamp experiments reveal that PnNs do not alter the bioelectrical properties of resting astrocytes, thus setting the scene for the use of PnN-loaded nanofibres as bioconductive platforms for interfacing astrocytes and controlling their bioelectrical properties., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Research over the past four decades has highlighted the importance of certain brain cells, called glial cells, and has moved the neurocentric vision of structure, function, and pathology of the nervous system toward a more holistic perspective. In this view, the demand for technologies that are able to target and both selectively monitor and control glial cells is emerging as a challenge across neuroscience, engineering, chemistry, and material science. Frequently neglected or marginally considered as a barrier to be overcome between neural implants and neuronal targets, glial cells, and in particular astrocytes, are increasingly considered as active players in determining the outcomes of device implantation. This review provides a concise overview not only of the previously established but also of the emerging physiological and pathological roles of astrocytes. It also critically discusses the most recent advances in biomaterial interfaces and devices that interact with glial cells and thus have enabled scientists to reach unprecedented insights into the role of astroglial cells in brain function and dysfunction. This work proposes glial interfaces and glial engineering as multidisciplinary fields that have the potential to enable significant advancement of knowledge surrounding cognitive function and acute and chronic neuropathologies., (© 2020 Wiley-VCH GmbH.)

Increasing evidences are demonstrating that structural and functional properties of non-neuronal brain cells, called astrocytes, such as those of cytoskeleton and of ion channels, are critical for brain physiology. Also, changes in astrocytes structure and function concur to and might determine the outcome of neuronal damage in acute neurological conditions or of chronic disease. Thus, the design and engineering of biomaterials that can drive the structural and functional properties of astrocytes is of growing interest for neuroregenerative medicine. Poly-ɛ-caprolactone (PCL), is FDA-approved polyester having excellent mechanical and chemical properties that can be tailored to obtain neural implants for regenerative purposes. However, the study on the use of PCL substrates for neuroregenerative purposes are mainly aimed at investigating the interaction of the material with neurons. Here, we report on the long-term viability, morphology, structural and functional properties of primary astrocytes grown on electrospun fibres of PCL (-GEL) and on blending of PCL and Gelatin protein (+GEL). We found that topography and morphological features of the substrate are the properties that mainly drives astrocytes adhesion and survival, over the long term, while they do not alter the cell function. Specifically, aligned PCL fibres induced in astrocytes a dramatic actin-cytoskeletal rearrangement as well as focal adhesion point number and distribution. Interestingly, structural changes observed in elongated astrocytes are not correlated with alterations in their electrophysiological properties. Our results indicated that PCL electrospun fibres are a permissive substrate that can be tuned to selectively alters astrocytes structural components while preserving astrocytes function. The results open the view for the use of PCL based electrospun fibres to target astrocytes for the treatment of brain dysfunction such as injuries or chronical disease., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Astrocytes are non-neuronal cells that govern the homeostatic regulation of the brain through ions and water transport, and Ca 2+ -mediated signaling. As they are tightly integrated into neural networks, label-free tools that can modulate cell function are needed to evaluate the role of astrocytes in brain physiology and dysfunction. Using live-cell fluorescence imaging, pharmacology, electrophysiology, and genetic manipulation, we show that pulsed infrared light can modulate astrocyte function through changes in intracellular Ca 2+ and water dynamics, providing unique mechanistic insight into the effect of pulsed infrared laser light on astroglial cells. Water transport is activated and, IP 3 R, TRPA1, TRPV4, and Aquaporin-4 are all involved in shaping the dynamics of infrared pulse-evoked intracellular calcium signal. These results demonstrate that astrocyte function can be modulated with infrared light. We expect that targeted control over calcium dynamics and water transport will help to study the crucial role of astrocytes in edema, ischemia, glioma progression, stroke, and epilepsy., (© 2020 Federation of American Societies for Experimental Biology.)

The correct human brain function is dependent on the activity of non-neuronal cells called astrocytes. The bioelectrical properties of astrocytes in vitro do not closely resemble those displayed in vivo and the former are incapable of generating action potential; thus, reliable approaches in vitro for noninvasive electrophysiological recording of astrocytes remain challenging for biomedical engineering. Here it is found that primary astrocytes grown on a device formed by a forest of randomly oriented gold coated-silicon nanowires, resembling the complex structural and functional phenotype expressed by astrocytes in vivo. The device enables noninvasive extracellular recording of the slow-frequency oscillations generated by differentiated astrocytes, while flat electrodes failed on recording signals from undifferentiated cells. Pathophysiological concentrations of extracellular potassium, occurring during epilepsy and spreading depression, modulate the power of slow oscillations generated by astrocytes. A reliable approach to study the role of astrocytes function in brain physiology and pathologies is presented., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Organic bioelectronics have a huge potential to generate interfaces and devices for the study of brain functions and for the therapy of brain pathologies. In this context, increasing efforts are needed to develop technologies for monitoring and stimulation of nonexcitable brain cells, called astrocytes. Astroglial calcium signaling plays, indeed, a pivotal role in the physiology and pathophysiology of the brain. Here, the use of transparent organic cell stimulating and sensing transistor (O-CST) architecture, fabricated with N,N'-ditridecylperylene-3,4,9,10-tetracarboxylic diimide (P13), to elicit and monitor intracellular calcium concentration ([Ca 2+ ] i ) in primary rat neocortical astrocytes is demonstrated. The transparency of O-CST allows performing calcium imaging experiments, showing that extracellular electrical stimulation of astrocytes induces a drastic increase in [Ca 2+ ] i . Pharmacological studies indicate that transient receptor potential (TRP) superfamily are critical mediators of the [Ca 2+ ] i increase. Experimental and computational analyses show that [Ca 2+ ] i response is enabled by the O-CST device architecture. Noteworthy, the extracellular field application induces a slight but significant increase in the cell volume. Collectively, it is shown that the O-CST is capable of selectively evoking astrocytes [Ca 2+ ] i , paving the way to the development of organic bioelectronic devices as glial interfaces to excite and control physiology of non-neuronal brain cells., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Consolidated evidence indicates that astroglial cells are critical in the homeostatic regulation of cellular volume by means of ion channels and aquaporin-4. Volume-regulated anion channel (VRAC) is the chloride channel that is activated upon cell swelling and critically contributes to cell volume regulation in astrocytes. The molecular identity of VRAC has been recently defined, revealing that it belongs to the leucine-rich repeat-containing 8 (LRRC8) protein family. However, there is a lack of evidence demonstrating that LRRC8A underpins VRAC currents in astrocyte. Nonetheless, direct evidence of the role of LRRC8A in astrocytic regulatory volume decrease remains to be proved. Here, we aim to bridge this gap in knowledge by combining RNA interference specific for LRRC8A with patch-clamp analyses and a water-permeability assay. We demonstrated that LRRC8A molecular expression is essential for swelling-activated chloride current via VRAC in primary-cultured cortical astrocytes. The knockdown of LRRC8A with a specific short interference RNA abolished the recovery of the cell volume after swelling induced by hypotonic challenge. In addition, immunoblotting, immunofluorescence, confocal imaging, and immunogold electron microscopy demonstrated that LRRC8A is expressed in the plasma membrane of primary cortical astrocytes and in situ in astrocytes at the perivascular interface with endothelial cells. Collectively, our results suggest that LRRC8A is an essential subunit of VRAC and a key factor for astroglial volume homeostasis.-Formaggio, F., Saracino, E., Mola, M. G., Rao, S. B., Amiry-Moghaddam, M., Muccini, M., Zamboni, R., Nicchia, G. P., Caprini, M., Benfenati, V. LRRC8A is essential for swelling-activated chloride current and for regulatory volume decrease in astrocytes.

Introduction: Cystic Echinococcosis (CE) is one of the most widespread zoonosis of veterinary and medical importance still constituting a sanitary, economic and socio-cultural problem in Italy., Methodology: The aim of this study was to update epidemiological data on cattle CE in Italy. Data on CE positivity of 5,336 cattle were acquired from abattoir registers between January 2009 and July 2010. Morphobiological characterization of hydatids was performed by direct examination of liver and lungs of 1,664 animals butchered in the same slaughterhouses in 2010. Strain typing of parasites was carried out through the amplification and sequencing of nd1 and cox1 mitochondrial genes., Results: Overall CE prevalence was of 8.1% (430/5,336). Parasitological examination of hydatids showed an overall prevalence of 8.6% with a fertility rate of 0.7% (12/1,664). Regarding localization, hydatids were found in 8% of the livers and in 7.6% of the lungs, respectively. Among positive animals, higher prevalence was observed in the liver (93%) compared to lungs (88.1%) (p > 0.05)., Conclusion: The economic loss due to organs condemnation related to CE in cattle amounted to almost € 24,000 per year in the examined abattoir during 2010. Sequence analysis showed the presence of G1 (sheep strain) or Echinococcus granulosus sensu strictu in all examined samples. The G1 confirmed, once more, its possible development into several intermediate hosts such as cattle, especially in areas like southern Italy and Sardinia where the lifecycle of the parasite is still to date carried on by sheep and dogs., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2017 Giovanni Poglayen, Antonio Varcasia, Anna Paola Pipia, Claudia Tamponi, Maria Parigi, Barbara Marchesi, Benedetto Morandi, Vannes Benfenati, Antonio Scala.)